Methods to identify soybean aphid resistant quantitative trait loci in soybean and compositions thereof

ABSTRACT

The present invention is in the field of plant breeding and aphid resistance. More specifically, the invention includes a method for breeding soybean plants containing quantitative trait loci that are associated with resistance to aphids,  Aphis glycines . The invention further includes method for monitoring the introgression quantitative trait loci (QTL) conferring aphid resistance into elite germplasm in a breeding program.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/963,936 filed Aug. 8, 2007. The entiretyof the application is hereby incorporated by reference.

INCORPORATION OF THE SEQUENCE LISTING

A sequence listing is contained in the file named “53776seqlisting.txt”which is 80,088 bytes (measured in MS-Windows) and was created on Aug.7, 2008. This electronic sequence listing is electronically filedherewith and is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of plant breeding. Morespecifically, the invention includes methods and compositions forscreening plants from the genus Glycine with markers associated withquantitative trait loci that are related to the aphid resistance inGlycine plants. The invention further includes methods and compositionsof genomic regions for screening plants from the genus Glycineassociated with aphid resistance.

BACKGROUND OF THE INVENTION

Soybean, Glycine max (L.) Merril, is a major economic crop worldwide andis a primary source of vegetable oil and protein (Sinclair and Backman,Compendium of Soybean Diseases, 3^(rd) Ed. APS Press, St. Paul, Minn.,p. 106. (1989). The growing demand for low cholesterol and high fiberdiets has also increased soybean's importance as a health food.

Soybean varieties grown in the United States have a narrow genetic base.Six introductions, ‘Mandarin,’ ‘Manchu,’ ‘Mandarin’ (Ottawa),“Richland,’ ‘AK’ (Harrow), and ‘Mukden,’ contributed nearly 70% of thegermplasm represented in 136 cultivar releases. To date, modern daycultivars can be traced back from these six soybean strains from China.In a study conducted by Cox et al., Crop Sci. 25:529-532 (1988), thesoybean germplasm is comprised of 90% adapted materials, 9% unadapted,and only 1% from exotic species. The genetic base of cultivated soybeancould be widened through exotic species. In addition, exotic species maypossess such key traits as disease, stress, and insect resistance.

Soybean aphid, Aphis glycines Matsumura, was identified as new insectpest of soybeans in 2001 and spread to over 21 states in the UnitedStates and 3 Canadian provinces by 2003 (Vennette et al. Ann Entomol SocAm 97:217-226 (2004)). High yields are critical to a farmer's profitmargin. Soybean aphid can cause over 50% yield losses (Wang et al.,Plant Protect 20:12-13 (1994)). In addition to the decrease in yield, anincrease in insecticide use can also decrease a farmer's profit margin.Over 7 million acres of soybean in the North Central U.S. were sprayedwith insecticide to control soybean aphids in 2003; the estimated costof the insecticide treatments was $84-$105 million in the North Centralregion alone in 2003 (Landis et al. NCR-125 Arthropod biologicalcontrol: state reports for 2003; Li et al., Mol Breeding 19:25-34(2007)).

Soybean aphids can directly damage the plant by removing significantamounts of water and nutrients causing the leaves to yellow and wilt.Additionally, aphids excrete honeydew, a sugar-rich sticky substance, onto the leaves and plants. Honeydew often leads to the development ofsooty mold, which affects photosynthesis resulting significant yieldlosses (Gomez et al., Environ Exp Bot 55: 77-86 (2006)). Soybean aphidsvector a number of viruses that can stunt plant growth, distorts leaves,cause mottling of leaves and stem, reduce pod number and causediscoloration in the seed. Viruses transmitted via soybean aphidinclude, Soybean mosaic virus, yellow mosaic virus, tobacco etch virusand tobacco vein mottling virus (Wang et al. Plant Dis 90: 920-926(2006)).

Host plant resistance to insect are often quantitatively inheritedtraits and not major resistance gene. Stacking quantitative resistancesis more durable than a major gene for resistance, but is difficult toidentify and incorporate multiple quantitative resistances into a singlesoybean variety. Molecular markers associated with insect resistanceoffers breeders a more efficient method to work with quantitative traitsand insect resistance. Aphid resistance genes and QTLs in soybean areknown. Examples of which including Rag1 was identified in the soybeanvariety Dowling and mapped to linkage group M (U.S. patent applicationSer. No. 11/158,307). Additionally, quantitative trait loci associatedwith aphid resistance were identified in Plant Introduction (PI) 567598Band mapped linkage groups B2, D1b, J and K (PCT/US2006/019200).

There is a need in the art of plant breeding to identify additionalquantitative trait loci associated with aphid resistance in soybean.Additionally, there is a need for rapid, cost-efficient method to assaythe absence or presence of aphid resistance loci in soybean. The presentinvention provides a method for screening and selecting a soybean plantcomprising a quantitative trait loci associated with aphid resistanceusing single nucleotide polymorphism (SNP) technology.

SUMMARY OF THE INVENTION

The present invention provides methods for producing aphid resistance insoybean plants. The present invention relates to methods to determinethe presence or absence of quantitative trait loci conferring aphidresistance in soybean plants, including but not limited to exoticgermplasm, populations, lines, elite lines, cultivars and varieties. Thepresent invention is not limited to any one type of aphid resistanttrait, such as antibiosis, antixenosis or repellency of aphids. Moreparticularly, the invention relates to methods involving for identifyingmolecular markers associated with aphid resistance quantitative traitloci (QTL). The present invention relates to the use of molecularmarkers to screen and select for aphid resistance within soybean plants,including but not limited to exotic germplasm, populations, lines, elitelines, and varieties.

In a preferred embodiment, the present invention further providesquantitative trait loci associated with resistance to one or more ofarthropods including but not limited to Coleoptera, examples of whichincluding Cerotoma sp. such as bean leaf beetle (Cerotoma trifurcata),Diabrotica sp. such as spotted cucumber beetle (Diabroticaundecimpunctata howardi), Epicauta sp. such as blister beetle (Epicautapestifera), Popilli sp. such as Japanese beetle (Popillia japonica),Dectes sp. such as soybean stem borer (Dectes texanus texanus), andColaspis sp. such as grape colaspis (Colaspis brunnea), etc.;Orthoptera, examples of which including Melanoplus sp. such asred-legged grasshopper (Melanoplus femurrubrum), and Shistocerca sp.such as American locust (Shistocerca Americana), etc.; Lepidoptera,examples of which including Plathypen sp. such as green cloverworm(Plathypena scabra), Pseudoplusia sp. such as soybean looper(Pseudoplusia includens), Anticarsia sp. such as velvetbean caterpillar(Anticarsia gemmatalis), Epargyreus sp. such as Silverspotted skipper(Epargyreus clarus), Estigmene sp. such as saltmarsh caterpillar(Estigmene acrea), Spodoptera sp. such as beet armyworm (Spodopteraexigua), Heliothis sp. such as Corn earworm (Heliothis zea), andMatsumuraeses sp. such as bean podworm (Matsumuraeses phaseoli);Hemiptera, examples of which including Acrosternum sp. such as greenstink bugs (Acrosternum hilare), Euschistus sp. such as brown stink bug(Euschistus servus), Nezara sp. such as southern stinkbug (Nezaraviridula); Homoptera, examples of which including Spissistilus sp. suchas threecornered alfalfa hopper (Spissistilus festinus), and Aphis sp.such as soybean aphid (Aphis glycines); Thysanoptera, examples of whichincluding Sericothrips sp. such as soybean thrips (Sericothripsvariabilis).

In a preferred embodiment, the present invention further provides lociassociated with resistance to nematodes, including, but not limited toHeterodera sp. such as soybean cyst nematode (Heterodera glycines),Belonolaimus sp. such as sting nematode (Belonolaimus longicaudatus),Rotylenchulus sp. such as reniform nematode (Rotylenchulus reniformis),Meloidogyne sp. such as southern root-knot nematode (Meloidogyneincognita), peanut root-knot nematode (Meloidogyne arenaria) and theJavanese root-knot nematode (Meloidogyne javanica).

The present invention relates to producing aphid resistant plants,populations, lines, elite lines, and varieties. More particularly, thepresent invention includes a method of introgressing an aphid resistantallele into a soybean plant comprising (A) crossing at least one firstsoybean plant comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 81 through SEQ ID NO: 120 with at least onesecond soybean plant in order to form a segregating population, (B)screening the segregating population with one or more nucleic acidmarkers to determine if one or more soybean plants from the segregatingpopulation contains the nucleic acid sequence, and (C) selecting fromthe segregation population one or more soybean plants comprising anucleic acid sequence selected from the group consisting of SEQ ID NO:81 through SEQ ID NO: 120.

The present invention includes a method of introgressing an allele intoa soybean plant comprising: (A) crossing at least one aphid resistantsoybean plant with at least one aphid susceptible soybean plant in orderto form a segregating population; (B) screening said segregatingpopulation with one or more nucleic acid markers to determine if one ormore soybean plants from said segregating population contains an aphidresistant allele, wherein said aphid resistance allele is an alleleselected from the group consisting of aphid resistance allele 1, aphidresistance allele 2, aphid resistance allele 3, aphid resistance allele4, aphid resistance allele 5, aphid resistance allele 6, aphidresistance allele 7, aphid resistance allele 8, aphid resistance allele9, aphid resistance allele 10, aphid resistance allele 11, aphidresistance allele 12, aphid resistance allele 13, aphid resistanceallele 14, aphid resistance allele 15, aphid resistance allele 16, aphidresistance allele 17, aphid resistance allele 18, aphid resistanceallele 19, aphid resistance allele 20, aphid resistance allele 21, aphidresistance allele 22, aphid resistance allele 23, aphid resistanceallele 24, aphid resistance allele 25, aphid resistance allele 26, aphidresistance allele 27, aphid resistance allele 28, aphid resistanceallele 29, aphid resistance allele 30, aphid resistance allele 31, aphidresistance allele 32, aphid resistance allele 33, aphid resistanceallele 34, aphid resistance allele 35, aphid resistance allele 36, aphidresistance allele 37 and aphid resistance allele 38, aphid resistanceallele 39, and aphid resistance allele 40.

The present invention includes an elite soybean plant comprising anucleic acid sequence selected from the group consisting of SEQ ID NO:81 through SEQ ID NO: 120.

BRIEF DESCRIPTION OF NUCLEIC ACID SEQUENCES

SEQ ID NO: 1 is a forward PCR primer for the amplification of SEQ ID NO:81.

SEQ ID NO: 2 is a reverse PCR primer for the amplification of SEQ ID NO:81.

SEQ ID NO: 3 is a forward PCR primer for the amplification of SEQ ID NO:82.

SEQ ID NO: 4 is a reverse PCR primer for the amplification of SEQ ID NO:82.

SEQ ID NO: 5 is a forward PCR primer for the amplification of SEQ ID NO:83.

SEQ ID NO: 6 is a reverse PCR primer for the amplification of SEQ ID NO:83.

SEQ ID NO: 7 is a forward PCR primer for the amplification of SEQ ID NO:84.

SEQ ID NO: 8 is a reverse PCR primer for the amplification of SEQ ID NO:84.

SEQ ID NO: 9 is a forward PCR primer for the amplification of SEQ ID NO:85.

SEQ ID NO: 10 is a reverse PCR primer for the amplification of SEQ IDNO: 85.

SEQ ID NO: 11 is a forward PCR primer for the amplification of SEQ IDNO: 86.

SEQ ID NO: 12 is a reverse PCR primer for the amplification of SEQ IDNO: 86.

SEQ ID NO: 13 is a forward PCR primer for the amplification of SEQ IDNO: 87.

SEQ ID NO: 14 is a reverse PCR primer for the amplification of SEQ IDNO: 87.

SEQ ID NO: 15 is a forward PCR primer for the amplification of SEQ IDNO: 88.

SEQ ID NO: 16 is a reverse PCR primer for the amplification of SEQ IDNO: 88.

SEQ ID NO: 17 is a forward PCR primer for the amplification of SEQ IDNO: 89.

SEQ ID NO: 18 is a reverse PCR primer for the amplification of SEQ IDNO: 89.

SEQ ID NO: 19 is a forward PCR primer for the amplification of SEQ IDNO: 90.

SEQ ID NO: 20 is a reverse PCR primer for the amplification of SEQ IDNO: 90.

SEQ ID NO: 21 is a forward PCR primer for the amplification of SEQ IDNO: 91.

SEQ ID NO: 22 is a reverse PCR primer for the amplification of SEQ IDNO: 91.

SEQ ID NO: 23 is a forward PCR primer for the amplification of SEQ IDNO: 92.

SEQ ID NO: 24 is a reverse PCR primer for the amplification of SEQ IDNO: 92.

SEQ ID NO: 25 is a forward PCR primer for the amplification of SEQ IDNO: 93.

SEQ ID NO: 26 is a reverse PCR primer for the amplification of SEQ IDNO: 93.

SEQ ID NO: 27 is a forward PCR primer for the amplification of SEQ IDNO: 94.

SEQ ID NO: 28 is a reverse PCR primer for the amplification of SEQ IDNO: 94.

SEQ ID NO: 29 is a forward PCR primer for the amplification of SEQ IDNO: 95.

SEQ ID NO: 30 is a reverse PCR primer for the amplification of SEQ IDNO: 95.

SEQ ID NO: 31 is a forward PCR primer for the amplification of SEQ IDNO: 96.

SEQ ID NO: 32 is a reverse PCR primer for the amplification of SEQ IDNO: 96.

SEQ ID NO: 33 is a forward PCR primer for the amplification of SEQ IDNO: 97.

SEQ ID NO: 34 is a reverse PCR primer for the amplification of SEQ IDNO: 97.

SEQ ID NO: 35 is a forward PCR primer for the amplification of SEQ IDNO: 98.

SEQ ID NO: 36 is a reverse PCR primer for the amplification of SEQ IDNO: 98.

SEQ ID NO: 37 is a forward PCR primer for the amplification of SEQ IDNO: 99.

SEQ ID NO: 38 is a reverse PCR primer for the amplification of SEQ IDNO: 99.

SEQ ID NO: 39 is a forward PCR primer for the amplification of SEQ IDNO: 100.

SEQ ID NO: 40 is a reverse PCR primer for the amplification of SEQ IDNO: 100.

SEQ ID NO: 41 is a forward PCR primer for the amplification of SEQ IDNO: 101.

SEQ ID NO: 42 is a reverse PCR primer for the amplification of SEQ IDNO: 101.

SEQ ID NO: 43 is a forward PCR primer for the amplification of SEQ IDNO: 102.

SEQ ID NO: 44 is a reverse PCR primer for the amplification of SEQ IDNO: 102.

SEQ ID NO: 45 is a forward PCR primer for the amplification of SEQ IDNO: 103.

SEQ ID NO: 46 is a reverse PCR primer for the amplification of SEQ IDNO: 103.

SEQ ID NO: 47 is a forward PCR primer for the amplification of SEQ IDNO: 104.

SEQ ID NO: 48 is a reverse PCR primer for the amplification of SEQ IDNO: 104.

SEQ ID NO: 49 is a forward PCR primer for the amplification of SEQ IDNO: 105.

SEQ ID NO: 50 is a reverse PCR primer for the amplification of SEQ IDNO: 105.

SEQ ID NO: 51 is a forward PCR primer for the amplification of SEQ IDNO: 106.

SEQ ID NO: 52 is a reverse PCR primer for the amplification of SEQ IDNO: 106.

SEQ ID NO: 53 is a forward PCR primer for the amplification of SEQ IDNO: 107.

SEQ ID NO: 54 is a reverse PCR primer for the amplification of SEQ IDNO: 107.

SEQ ID NO: 55 is a forward PCR primer for the amplification of SEQ IDNO: 108.

SEQ ID NO: 56 is a reverse PCR primer for the amplification of SEQ IDNO: 108;

SEQ ID NO: 57 is a forward PCR primer for the amplification of SEQ IDNO: 109.

SEQ ID NO: 58 is a reverse PCR primer for the amplification of SEQ IDNO: 109.

SEQ ID NO: 59 is a forward PCR primer for the amplification of SEQ IDNO: 110.

SEQ ID NO: 60 is a reverse PCR primer for the amplification of SEQ IDNO: 110.

SEQ ID NO: 61 is a forward PCR primer for the amplification of SEQ IDNO: 111.

SEQ ID NO: 62 is a reverse PCR primer for the amplification of SEQ IDNO: 111

SEQ ID NO: 63 is a forward PCR primer for the amplification of SEQ IDNO: 112.

SEQ ID NO: 64 is a reverse PCR primer for the amplification of SEQ IDNO: 112.

SEQ ID NO: 65 is a forward PCR primer for the amplification of SEQ IDNO: 113.

SEQ ID NO: 66 is a reverse PCR primer for the amplification of SEQ IDNO: 113

SEQ ID NO: 67 is a forward PCR primer for the amplification of SEQ IDNO: 114.

SEQ ID NO: 68 is a reverse PCR primer for the amplification of SEQ IDNO: 114.

SEQ ID NO: 69 is a forward PCR primer for the amplification of SEQ IDNO: 115.

SEQ ID NO: 70 is a reverse PCR primer for the amplification of SEQ IDNO: 115.

SEQ ID NO: 71 is a forward PCR primer for the amplification of SEQ IDNO: 116.

SEQ ID NO: 72 is a reverse PCR primer for the amplification of SEQ IDNO: 116.

SEQ ID NO: 73 is a forward PCR primer for the amplification of SEQ IDNO: 117.

SEQ ID NO: 74 is a reverse PCR primer for the amplification of SEQ IDNO: 117.

SEQ ID NO: 75 is a forward PCR primer for the amplification of SEQ IDNO: 118.

SEQ ID NO: 76 is a reverse PCR primer for the amplification of SEQ IDNO: 118.

SEQ ID NO: 77 is a forward PCR primer for the amplification of SEQ IDNO: 119.

SEQ ID NO: 78 is a reverse PCR primer for the amplification of SEQ IDNO: 119.

SEQ ID NO: 79 is a forward PCR primer for the amplification of SEQ IDNO: 120.

SEQ ID NO: 80 is a reverse PCR primer for the amplification of SEQ IDNO: 120.

SEQ ID NO: 81 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 1.

SEQ ID NO: 82 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 1.

SEQ ID NO: 83 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 1.

SEQ ID NO: 84 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 1.

SEQ ID NO: 85 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 2.

SEQ ID NO: 86 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 3.

SEQ ID NO: 87 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 4.

SEQ ID NO: 88 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 5.

SEQ ID NO: 89 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 6.

SEQ ID NO: 90 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 7.

SEQ ID NO: 91 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 8.

SEQ ID NO: 92 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 8.

SEQ ID NO: 93 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 9.

SEQ ID NO: 94 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 10.

SEQ ID NO: 95 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 11.

SEQ ID NO: 96 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 12.

SEQ ID NO: 97 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 13.

SEQ ID NO: 98 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 14.

SEQ ID NO: 99 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 15.

SEQ ID NO: 100 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 16.

SEQ ID NO: 101 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 16.

SEQ ID NO: 102 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 16.

SEQ ID NO: 103 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 17.

SEQ ID NO: 104 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 18.

SEQ ID NO: 105 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 19.

SEQ ID NO: 106 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 20.

SEQ ID NO: 107 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 21.

SEQ ID NO: 108 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 21.

SEQ ID NO: 109 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 22.

SEQ ID NO: 110 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 23.

SEQ ID NO: 111 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 23.

SEQ ID NO: 112 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 24.

SEQ ID NO: 113 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 25.

SEQ ID NO: 114 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 26.

SEQ ID NO: 115 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 26.

SEQ ID NO: 116 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 27.

SEQ ID NO: 117 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 28.

SEQ ID NO: 118 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 28.

SEQ ID NO: 119 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 28.

SEQ ID NO: 120 is a genomic sequence derived from Glycine Maxcorresponding to aphid resistance locus 28.

SEQ ID NO: 121 is a probe for the detection of the SNP of SEQ ID NO: 81.

SEQ ID NO: 122 is a probe for the detection of the SNP of SEQ ID NO: 81.

SEQ ID NO: 123 is a probe for the detection of the SNP of SEQ ID NO: 82.

SEQ ID NO: 124 is a probe for the detection of the SNP of SEQ ID NO: 82.

SEQ ID NO: 125 is a probe for the detection of the SNP of SEQ ID NO: 83.

SEQ ID NO: 126 is a probe for the detection of the SNP of SEQ ID NO: 83.

SEQ ID NO: 127 is a probe for the detection of the SNP of SEQ ID NO: 84.

SEQ ID NO: 128 is a probe for the detection of the SNP of SEQ ID NO: 84.

SEQ ID NO: 129 is a probe for the detection of the SNP of SEQ ID NO: 85.

SEQ ID NO: 130 is a probe for the detection of the SNP of SEQ ID NO: 85.

SEQ ID NO: 131 is a probe for the detection of the SNP of SEQ ID NO: 86.

SEQ ID NO: 132 is a probe for the detection of the SNP of SEQ ID NO: 86.

SEQ ID NO: 133 is a probe for the detection of the SNP of SEQ ID NO: 87.

SEQ ID NO: 134 is a probe for the detection of the SNP of SEQ ID NO: 87.

SEQ ID NO: 135 is a probe for the detection of the SNP of SEQ ID NO: 88.

SEQ ID NO: 136 is a probe for the detection of the SNP of SEQ ID NO: 88.

SEQ ID NO: 137 is a probe for the detection of the SNP of SEQ ID NO: 89.

SEQ ID NO: 138 is a probe for the detection of the SNP of SEQ ID NO: 89.

SEQ ID NO: 139 is a probe for the detection of the SNP of SEQ ID NO: 90.

SEQ ID NO: 140 is a probe for the detection of the SNP of SEQ ID NO: 90.

SEQ ID NO: 141 is a probe for the detection of the SNP of SEQ ID NO: 91.

SEQ ID NO: 142 is a probe for the detection of the SNP of SEQ ID NO: 91.

SEQ ID NO: 143 is a probe for the detection of the SNP of SEQ ID NO: 92.

SEQ ID NO: 144 is a probe for the detection of the SNP of SEQ ID NO: 92.

SEQ ID NO: 145 is a probe for the detection of the SNP of SEQ ID NO: 93.

SEQ ID NO: 146 is a probe for the detection of the SNP of SEQ ID NO: 93.

SEQ ID NO: 147 is a probe for the detection of the SNP of SEQ ID NO: 94.

SEQ ID NO: 148 is a probe for the detection of the SNP of SEQ ID NO: 94.

SEQ ID NO: 149 is a probe for the detection of the SNP of SEQ ID NO: 95.

SEQ ID NO: 150 is a probe for the detection of the SNP of SEQ ID NO: 95.

SEQ ID NO: 151 is a probe for the detection of the SNP of SEQ ID NO: 96.

SEQ ID NO: 152 is a probe for the detection of the SNP of SEQ ID NO: 96.

SEQ ID NO: 153 is a probe for the detection of the SNP of SEQ ID NO: 97.

SEQ ID NO: 154 is a probe for the detection of the SNP of SEQ ID NO: 97.

SEQ ID NO: 155 is a probe for the detection of the SNP of SEQ ID NO: 98.

SEQ ID NO: 156 is a probe for the detection of the SNP of SEQ ID NO: 98.

SEQ ID NO: 157 is a probe for the detection of the SNP of SEQ ID NO: 99.

SEQ ID NO: 158 is a probe for the detection of the SNP of SEQ ID NO: 99.

SEQ ID NO: 159 is a probe for the detection of the SNP of SEQ ID NO:100.

SEQ ID NO: 160 is a probe for the detection of the SNP of SEQ ID NO:100.

SEQ ID NO: 161 is a probe for the detection of the SNP of SEQ ID NO:101.

SEQ ID NO: 162 is a probe for the detection of the SNP of SEQ ID NO:101.

SEQ ID NO: 163 is a probe for the detection of the SNP of SEQ ID NO:102.

SEQ ID NO: 164 is a probe for the detection of the SNP of SEQ ID NO:102.

SEQ ID NO: 165 is a probe for the detection of the SNP of SEQ ID NO:103.

SEQ ID NO: 166 is a probe for the detection of the SNP of SEQ ID NO:103.

SEQ ID NO: 167 is a probe for the detection of the SNP of SEQ ID NO:104.

SEQ ID NO: 168 is a probe for the detection of the SNP of SEQ ID NO:104.

SEQ ID NO: 169 is a probe for the detection of the SNP of SEQ ID NO:105.

SEQ ID NO: 170 is a probe for the detection of the SNP of SEQ ID NO:105.

SEQ ID NO: 171 is a probe for the detection of the SNP of SEQ ID NO:106.

SEQ ID NO: 172 is a probe for the detection of the SNP of SEQ ID NO:106.

SEQ ID NO: 173 is a probe for the detection of the SNP of SEQ ID NO:107.

SEQ ID NO: 174 is a probe for the detection of the SNP of SEQ ID NO:107.

SEQ ID NO: 175 is a probe for the detection of the SNP of SEQ ID NO:108.

SEQ ID NO: 176 is a probe for the detection of the SNP of SEQ ID NO:108.

SEQ ID NO: 177 is a probe for the detection of the SNP of SEQ ID NO:109.

SEQ ID NO: 178 is a probe for the detection of the SNP of SEQ ID NO:109.

SEQ ID NO: 179 is a probe for the detection of the SNP of SEQ ID NO:110.

SEQ ID NO: 180 is a probe for the detection of the SNP of SEQ ID NO:110.

SEQ ID NO: 181 is a probe for the detection of the SNP of SEQ ID NO:111.

SEQ ID NO: 182 is a probe for the detection of the SNP of SEQ ID NO:111.

SEQ ID NO: 183 is a probe for the detection of the SNP of SEQ ID NO:112.

SEQ ID NO: 184 is a probe for the detection of the SNP of SEQ ID NO:112.

SEQ ID NO: 185 is a probe for the detection of the SNP of SEQ ID NO:113.

SEQ ID NO: 186 is a probe for the detection of the SNP of SEQ ID NO:113.

SEQ ID NO: 187 is a probe for the detection of the SNP of SEQ ID NO:114.

SEQ ID NO: 188 is a probe for the detection of the SNP of SEQ ID NO:114.

SEQ ID NO: 189 is a probe for the detection of the SNP of SEQ ID NO:115.

SEQ ID NO: 190 is a probe for the detection of the SNP of SEQ ID NO:115.

SEQ ID NO: 191 is a probe for the detection of the SNP of SEQ ID NO:116.

SEQ ID NO: 192 is a probe for the detection of the SNP of SEQ ID NO:116.

SEQ ID NO: 193 is a probe for the detection of the SNP of SEQ ID NO:117.

SEQ ID NO: 194 is a probe for the detection of the SNP of SEQ ID NO:117.

SEQ ID NO: 195 is a probe for the detection of the SNP of SEQ ID NO:118.

SEQ ID NO: 196 is a probe for the detection of the SNP of SEQ ID NO:118.

SEQ ID NO: 197 is a probe for the detection of the SNP of SEQ ID NO:119.

SEQ ID NO: 198 is a probe for the detection of the SNP of SEQ ID NO:119.

SEQ ID NO: 199 is a probe for the detection of the SNP of SEQ ID NO:120.

SEQ ID NO: 200 is a probe for the detection of the SNP of SEQ ID NO:120.

SEQ ID NO: 201 is a probe for the detection of the SNP of SEQ ID NO: 82.

SEQ ID NO: 202 is a probe for the detection of the SNP of SEQ ID NO: 82.

SEQ ID NO: 203 is a probe for the detection of the SNP of SEQ ID NO: 83.

SEQ ID NO: 204 is a probe for the detection of the SNP of SEQ ID NO: 83.

SEQ ID NO: 205 is a probe for the detection of the SNP of SEQ ID NO: 84.

SEQ ID NO: 206 is a probe for the detection of the SNP of SEQ ID NO: 84.

SEQ ID NO: 207 is a probe for the detection of the SNP of SEQ ID NO:100.

SEQ ID NO: 208 is a probe for the detection of the SNP of SEQ ID NO:100.

SEQ ID NO: 209 is a probe for the detection of the SNP of SEQ ID NO:111.

SEQ ID NO: 210 is a probe for the detection of the SNP of SEQ ID NO:111.

SEQ ID NO: 211 is a probe for the detection of the SNP of SEQ ID NO:117.

SEQ ID NO: 212 is a probe for the detection of the SNP of SEQ ID NO:117.

SEQ ID NO: 213 is a probe for the detection of the SNP of SEQ ID NO:119.

SEQ ID NO: 214 is a probe for the detection of the SNP of SEQ ID NO:119.

SEQ ID NO: 215 is a probe for the detection of the SNP of SEQ ID NO:120.

SEQ ID NO: 216 is a probe for the detection of the SNP of SEQ ID NO:120.

SEQ ID NO: 217 is a probe for the detection of the SNP of SEQ ID NO: 82.

SEQ ID NO: 218 is a probe for the detection of the SNP of SEQ ID NO: 83.

SEQ ID NO: 219 is a probe for the detection of the SNP of SEQ ID NO: 84.

SEQ ID NO: 220 is a probe for the detection of the SNP of SEQ ID NO:100.

SEQ ID NO: 221 is a probe for the detection of the SNP of SEQ ID NO:111.

SEQ ID NO: 222 is a probe for the detection of the SNP of SEQ ID NO:117.

SEQ ID NO: 223 is a probe for the detection of the SNP of SEQ ID NO:119.

SEQ ID NO: 224 is a probe for the detection of the SNP of SEQ ID NO:120.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides 28 aphid resistance loci that are locatedon linkage group A1, B1, B2, C1, D1a, D1b, E, F, G, H, I, and O in thesoybean genome that are not previously associated with associated withaphid resistance (Table 1). The present invention also provides forquantitative trait loci (QTL) alleles capable of conferring resistanceto soybean aphid. Alleles that are located at aphid resistance locus 1,aphid resistance locus 2, aphid resistance locus 3, aphid resistancelocus 4, aphid resistance locus 5, aphid resistance locus 6, aphidresistance locus 7, aphid resistance locus 8, aphid resistance locus 9,aphid resistance locus 10, aphid resistance locus 11, aphid resistancelocus 12, aphid resistance locus 13, aphid resistance locus 14, aphidresistance locus 15, aphid resistance locus 16, aphid resistance locus17, aphid resistance locus 18, aphid resistance locus 19, aphidresistance locus 20, aphid resistance locus 21, aphid resistance locus22, aphid resistance locus 23, aphid resistance locus 24, aphidresistance locus 25, aphid resistance locus 26, aphid resistance locus27, and aphid resistance locus 28 are provided.

In the present invention, aphid resistance locus 1 is located on linkagegroup J. SNP markers used to monitor the introgression of aphidresistance locus 1 are SEQ ID NO: 81 through SEQ ID NO: 84. SNP markerDNA sequences associated with aphid resistance locus 1 (SEQ ID NO: 81through SEQ ID NO: 84) can be amplified using the primers indicated asSEQ ID NO: 1 through SEQ ID NO: 8 and detected with probes indicated asSEQ ID NO: 121 through SEQ ID NO: 128, SEQ ID NO: 201 through SEQ ID NO:206, and SEQ ID NO: 217 through SEQ ID NO: 219.

In the present invention, aphid resistance locus 2 is located on linkagegroup E. SNP marker used to monitor the introgression of aphidresistance locus 2 is SEQ ID NO: 85. SNP marker DNA sequences associatedwith aphid resistance locus 2 (SEQ ID NO: 85) can be amplified using theprimers indicated as SEQ ID NO: 9 through SEQ ID NO: 10 and detectedwith probes indicated as SEQ ID NO: 129 through SEQ ID NO: 130.

In the present invention, aphid resistance locus 3 is located on linkagegroup E. SNP marker used to monitor the introgression of aphidresistance locus 3 is SEQ ID NO: 86. SNP marker DNA sequences associatedwith aphid resistance locus 3 (SEQ ID NO: 86) can be amplified using theprimers indicated as SEQ ID NO: 11 through SEQ ID NO: 12 and detectedwith probes indicated as SEQ ID NO: 131 through SEQ ID NO: 132.

In the present invention, aphid resistance locus 4 is located on linkagegroup E. SNP marker used to monitor the introgression of aphidresistance locus 4 is SEQ ID NO: 87. SNP marker DNA sequences associatedwith aphid resistance locus 4 (SEQ ID NO: 87) can be amplified using theprimers indicated as SEQ ID NO: 13 through SEQ ID NO: 14 and detectedwith probes indicated as SEQ ID NO: 133 through SEQ ID NO: 134.

In the present invention, aphid resistance locus 5 is located on linkagegroup B1. SNP marker used to monitor the introgression of aphidresistance locus 5 is SEQ ID NO: 88. SNP marker DNA sequences associatedwith aphid resistance locus 5 (SEQ ID NO: 88) can be amplified using theprimers indicated as SEQ ID NO: 15 through SEQ ID NO: 16 and detectedwith probes indicated as SEQ ID NO: 135 through SEQ ID NO: 136.

In the present invention, aphid resistance locus 6 is located on linkagegroup N. SNP marker used to monitor the introgression of aphidresistance locus 6 is SEQ ID NO: 89. SNP marker DNA sequences associatedwith aphid resistance locus 6 (SEQ ID NO: 89) can be amplified using theprimers indicated as SEQ ID NO: 17 through SEQ ID NO: 18 and detectedwith probes indicated as SEQ ID NO: 137 through SEQ ID NO: 138.

In the present invention, aphid resistance locus 7 is located on linkagegroup G. SNP marker used to monitor the introgression of aphidresistance locus 7 is SEQ ID NO: 90. SNP marker DNA sequences associatedwith aphid resistance locus 7 (SEQ ID NO: 90) can be amplified using theprimers indicated as SEQ ID NO: 19 through SEQ ID NO: 20 and detectedwith probes indicated as SEQ ID NO: 139 through SEQ ID NO: 140.

In the present invention, aphid resistance locus 8 is located on linkagegroup N. SNP markers used to monitor the introgression of aphidresistance locus 8 are SEQ ID NO: 91 through SEQ ID NO 92. SNP markerDNA sequences associated with aphid resistance locus 8 (91 through SEQID NO 92) can be amplified using the primers indicated as SEQ ID NO: 21through SEQ ID NO: 24 and detected with probes indicated as SEQ ID NO:141 through SEQ ID NO: 144.

In the present invention, aphid resistance locus 9 is located on linkagegroup N. SNP marker used to monitor the introgression of aphidresistance locus 9 is SEQ ID NO: 93. SNP marker DNA sequences associatedwith aphid resistance locus 9 (SEQ ID NO: 93) can be amplified using theprimers indicated as SEQ ID NO: 25 through SEQ ID NO: 26 and detectedwith probes indicated as SEQ ID NO: 145 through SEQ ID NO: 146.

In the present invention, aphid resistance locus 10 is located onlinkage group A1. SNP marker used to monitor the introgression of aphidresistance locus 10 is SEQ ID NO: 94. SNP marker DNA sequencesassociated with aphid resistance locus 10 (SEQ ID NO: 94) can beamplified using the primers indicated as SEQ ID NO: 27 through SEQ IDNO: 28 and detected with probes indicated as SEQ ID NO: 147 through SEQID NO: 148.

In the present invention, aphid resistance locus 11 is located onlinkage group A1. SNP marker used to monitor the introgression of aphidresistance locus 11 is SEQ ID NO: 95. SNP marker DNA sequencesassociated with aphid resistance locus 11 (SEQ ID NO: 95) can beamplified using the primers indicated as SEQ ID NO: 29 through SEQ IDNO: 30 and detected with probes indicated as SEQ ID NO: 149 through SEQID NO: 150.

In the present invention, aphid resistance locus 12 is located onlinkage group DIa. SNP marker used to monitor the introgression of aphidresistance locus 12 is SEQ ID NO: 96. SNP marker DNA sequencesassociated with aphid resistance locus 12 (SEQ ID NO: 96) can beamplified using the primers indicated as SEQ ID NO: 31 through SEQ IDNO: 32 and detected with probes indicated as SEQ ID NO: 151 through SEQID NO: 152.

In the present invention, aphid resistance locus 13 is located onlinkage group C2. SNP marker used to monitor the introgression of aphidresistance locus 13 is SEQ ID NO: 97. SNP marker DNA sequencesassociated with aphid resistance locus 13 (SEQ ID NO: 97) can beamplified using the primers indicated as SEQ iD NO: 33 through SEQ IDNO: 34 and detected with probes indicated as SEQ ID NO: 153 through SEQID NO: 154.

In the present invention, aphid resistance locus 14 is located onlinkage group H. SNP marker used to monitor the introgression of aphidresistance locus 14 is SEQ ID NO: 98. SNP marker DNA sequencesassociated with aphid resistance locus 14 (SEQ ID NO: 98) can beamplified using the primers indicated as SEQ ID NO: 35 through SEQ IDNO: 36 and detected with probes indicated as SEQ ID NO: 155 through SEQID NO: 156.

In the present invention, aphid resistance locus 15 is located onlinkage group H. SNP marker used to monitor the introgression of aphidresistance locus 15 is SEQ ID NO: 99. SNP marker DNA sequencesassociated with aphid resistance locus 15 (SEQ ID NO: 99) can beamplified using the primers indicated as SEQ ID NO: 37 through SEQ IDNO: 38 and detected with probes indicated as SEQ ID NO: 157 through SEQID NO: 158.

In the present invention, aphid resistance locus 16 is located onlinkage group D2. SNP marker used to monitor the introgression of aphidresistance locus 16 is SEQ ID NO: 100 through SEQ ID NO: 102. SNP markerDNA sequences associated with aphid resistance locus 16 (SEQ ID NO: 100through SEQ ID NO: 102) can be amplified using the primers indicated asSEQ ID NO: 39 through SEQ ID NO: 44 and detected with probes indicatedas SEQ ID NO: 159 through SEQ ID NO: 162, SEQ ID NO: 207 through SEQ IDNO: 208, and SEQ ID NO: 220.

In the present invention, aphid resistance locus 17 is located onlinkage group F. SNP marker used to monitor the introgression of aphidresistance locus 17 is SEQ ID NO: 103. SNP marker DNA sequencesassociated with aphid resistance locus 17 (SEQ ID NO: 103) can beamplified using the primers indicated as SEQ ID NO: 45 through SEQ IDNO: 46 and detected with probes indicated as SEQ ID NO: 165 through SEQID NO: 166.

In the present invention, aphid resistance locus 18 is located onlinkage group F. SNP marker used to monitor the introgression of aphidresistance locus 18 is SEQ ID NO: 104. SNP marker DNA sequencesassociated with aphid resistance locus 18 (SEQ ID NO: 104) can beamplified using the primers indicated as SEQ ID NO: 47 through SEQ IDNO: 48 and detected with probes indicated as SEQ ID NO: 167 through SEQID NO: 168.

In the present invention, aphid resistance locus 19 is located onlinkage group I. SNP marker used to monitor the introgression of aphidresistance locus 19 is SEQ ID NO: 105. SNP marker DNA sequencesassociated with aphid resistance locus 19 (SEQ ID NO: 105) can beamplified using the primers indicated as SEQ ID NO: 49 through SEQ IDNO: 50 and detected with probes indicated as SEQ ID NO: 169 through SEQID NO: 170.

In the present invention, aphid resistance locus 20 is located onlinkage group D1b. SNP marker used to monitor the introgression of aphidresistance locus 20 is SEQ ID NO: 106. SNP marker DNA sequencesassociated with aphid resistance locus 20 (SEQ ID NO: 106) can beamplified using the primers indicated as SEQ ID NO: 51 through SEQ IDNO: 52 and detected with probes indicated as SEQ ID NO: 171 through SEQID NO: 172.

In the present invention, aphid resistance locus 21 is located onlinkage group D1b. SNP markers used to monitor the introgression ofaphid resistance locus 21 are SEQ ID NO: 107 through SEQ ID NO. 108. SNPmarkers DNA sequences associated with aphid resistance locus 21 (SEQ IDNO: 107 through SEQ ID NO. 108) can be amplified using the primersindicated as SEQ ID NO: 53 through SEQ ID NO: 56 and detected withprobes indicated as SEQ ID NO: 173 through SEQ ID NO: 176.

In the present invention, aphid resistance locus 22 is located onlinkage group O, SNP marker used to monitor the introgression of aphidresistance locus 22 is SEQ ID NO: 109. SNP marker DNA sequencesassociated with aphid resistance locus 22 (SEQ ID NO: 109) can beamplified using the primers indicated as SEQ ID NO: 57 through SEQ IDNO: 58 and detected with probes indicated as SEQ ID NO: 177 through SEQID NO: 178.

In the present invention, aphid resistance locus 23 is located onlinkage group O, SNP markers used to monitor the introgression of aphidresistance locus 23 are SEQ ID NO: 110 through SEQ ID NO: 111. SNPmarker DNA sequences associated with aphid resistance locus 23 (SEQ IDNO: 110 through SEQ ID NO: 111) can be amplified using the primersindicated as SEQ ID NO: 59 through SEQ ID NO: 62 and detected withprobes indicated as SEQ ID NO: 179 through SEQ ID NO: 182, SEQ ID NO:209 through SEQ ID NO: 210, and SEQ ID NO: 221.

In the present invention, aphid resistance locus 24 is located onlinkage group C1. SNP marker used to monitor the introgression of aphidresistance locus 24 is SEQ ID NO: 112. SNP marker DNA sequencesassociated with aphid resistance locus 24 (SEQ ID NO: 112) can beamplified using the primers indicated as SEQ ID NO: 63 through SEQ IDNO: 64 and detected with probes indicated as SEQ ID NO: 183 through SEQID NO: 184.

In the present invention, aphid resistance locus 25 is located onlinkage group C1. SNP marker used to monitor the introgression of aphidresistance locus 25 is SEQ ID NO: 113. SNP marker DNA sequencesassociated with aphid resistance locus 25 (SEQ ID NO: 113) can beamplified using the primers indicated as SEQ ID NO: 65 through SEQ IDNO: 66 and detected with probes indicated as SEQ ID NO: 185 through SEQID NO: 186.

In the present invention, aphid resistance locus 26 is located onlinkage group C1. SNP markers used to monitor the introgression of aphidresistance locus 26 are SEQ ID NO: 114 through SEQ ID NO: 115. SNPmarker DNA sequences associated with aphid resistance locus 26 (SEQ IDNO: 114 through SEQ ID NO: 115) can be amplified using the primersindicated as SEQ ID NO: 67 through SEQ ID NO: 70 and detected withprobes indicated as SEQ ID NO: 187 through SEQ ID NO: 190.

In the present invention, aphid resistance locus 27 is located onlinkage group K. SNP marker used to monitor the introgression of aphidresistance locus 27 is SEQ ID NO: 116. SNP marker DNA sequencesassociated with aphid resistance locus 27 (SEQ ID NO: 116) can beamplified using the primers indicated as SEQ ID NO: 71 through SEQ IDNO: 72 and detected with probes indicated as SEQ ID NO: 191 through SEQID NO: 192.

In the present invention, aphid resistance locus 28 is located onlinkage group B2. SNP markers used to monitor the introgression of aphidresistance locus 28 are SEQ ID NO: 117 through SEQ ID NO: 120. SNPmarker DNA sequences associated with aphid resistance locus 28 (SEQ IDNO: 117 through SEQ ID NO: 120) can be amplified using the primersindicated as SEQ ID NO: 73 through SEQ ID NO: 80 and detected withprobes indicated as SEQ ID NO: 193 through SEQ ID NO: 200, SEQ ID NO:211-216, and SEQ ID NO: 222-223.

The present invention also provides a soybean plant comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO: 81through SEQ ID NO: 120 and complements thereof. In one aspect, thesoybean plant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleic acidsequences selected from the group consisting of SEQ ID NO: 81 throughSEQ ID NO: 120, fragment thereof, and complements thereof.

The present invention also provides a soybean plant comprising 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 aphid resistance loci where one or more alleles atone or more of their loci are selected from the group consisting ofaphid resistance allele 1, aphid resistance allele 2, aphid resistanceallele 3, aphid resistance allele 4, aphid resistance allele 5, aphidresistance allele 6, aphid resistance allele 7, aphid resistance allele8, aphid resistance allele 9, aphid resistance allele 10, aphidresistance allele 11, aphid resistance allele 12, aphid resistanceallele 13, aphid resistance allele 14, aphid resistance allele 15, aphidresistance allele 16, aphid resistance allele 17, aphid resistanceallele 18, aphid resistance allele 19, aphid resistance allele 20, aphidresistance allele 21, aphid resistance allele 22, aphid resistanceallele 23, aphid resistance allele 24, aphid resistance allele 25, aphidresistance allele 26, aphid resistance allele 27, aphid resistanceallele 28, aphid resistance allele 29, aphid resistance allele 30, aphidresistance allele 31, aphid resistance allele 32, aphid resistanceallele 33, aphid resistance allele 34, aphid resistance allele 35, aphidresistance allele 36, aphid resistance allele 37, aphid resistanceallele 38, aphid resistance allele 39, and aphid resistance allele 40.Such alleles may be homozygous or heterozygous.

As used herein, aphid refers to any of various small, soft-bodied,plant-sucking insects of the Order Homoptera, further of the familyAphididae, wherein examples of Aphididae include but are not limited tothe genus of Acyrthosiphon, Allocotaphis, Amphorophora, Anoecia,Anuraphis, Aphidounguis, Aphidura, Aphis, Asiphonaphis, Astegopteryx,Aulacorthum, Betacallis, Betulaphis, Boernerina, Brachycaudus,Brachycorynella, Brevicoryne, Calaphis, Callipterinella, Callipterus,Cavariella, Cerataphis, Ceratovacuna, Chaetomyzus, Chaetosiphon,Chaitophorus, Chaitoregma, Chromaphis, Cinara, Clethrobius,Clydesmithia, Coloradoa, Cornaphis, Cryptomyzus, Crypturaphis, Doralis,Doraphis, Drepanaphis, Drepanosiphoniella, Drepanosiphum, Dysaphis,Eomacrosiphum, Epipemphigus, Ericolophium, Eriosoma, Essigella,Euceraphis, Eulachnus, Eumyzus, Eutrichosiphum, Fimbriaphis, Fullawaya,Geopemphigus, Glyphina, Gootiella, Greenidea, Grylloprociphilus,Hamamelistes, Hannabura, Hornaphis, Hyadaphis, Hyalomyzus, Hyalopterus,Hyperomyza, Hyperomyzus, Hysteroneura, Illinoia, Indiaphis,Indomasonaphis, Kakimia, Lachnus, Laingia, Lambersaphis, Latgerina,Longicaudus, Longistigma, Macromyzus, Macrosiphoniella, etc. while evenfurther any one or more of the following genus species of Aphididae,examples of which including soybean aphid Aphis glycines, Bean aphidAphis fabae, Cotton aphid Aphis gossypii, Rose aphid Macrosiphun rosae,green peach aphid Myzus persicae, corn leaf aphid Rhopalosiphum maidis,spotted alfalfa aphid Therioaphis maculata, wooly apple aphid Eriosomalanigerum and the like.

As used herein, soybean aphid, Aphis glycines, and Aphis glycinesMatasamura refers to an aphid that feeds on soybean. However, any aphidthat is found on and feeds on a soybean plant, such as the bean aphisAphis fabae is a target for aphid resistance in soybean and is withinthe scope of the invention. A soybean plant of the present invention canbe resistant to one or more aphids infesting a soybean plant. In oneaspect, the present invention provides plants resistant to aphids aswell as methods and compositions for screening soybean plants forresistance or, susceptibility to aphids, caused by the genus Aphis. In apreferred aspect, the present invention provides methods andcompositions for screening soybean plants for resistance orsusceptibility to Aphis glycines.

In an aspect, the plant is selected from the genus Glycine. Glycineplants, including but not limited to exotic germplasm, populations,lines, elite lines, cultivars and varieties.

As uses herein, soybean plant refers to a plant of the family Fabaceae,herein uses in the broadest sense and includes but is not limited to anyspecies of soybean, examples of which including a Glycine species. Asoybean plant may be a Glycine arenaria, Glycine argyrea, Glycinecanescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba,Glycinefalcate, Glycine latifolia, Glycine latrobeana, Glycine max,Glycine microphylla, Glycine pescadrensis, Glycine pindanica, Glycinerubiginosa, Glycine soja, Glycine sp., Glycine stenophita, Glycinetabacina, and Glycine tomentella.

Plants of the present invention can be a soybean plant that is veryresistant, resistant, substantially resistant, mid-resistant,comparatively resistant, partially resistant, mid-susceptible, orsusceptible.

As used herein, the term resistant, resistance and host plant resistancerefers the ability of a host plant to prevent or reduce infestation anddamage of a pest from the group comprising insects, nematodes,pathogens, fungi, viruses, and diseases.

As used herein, the term antixenosis or non-preference resistance refersto the ability of a plant to ability to repel insects, causing areduction in egg laying and feeding.

As used herein, the term antibiosis refers the ability of a plant toreduce survival, growth, or reproduction of insects that feed on it.

As used herein, the term tolerance refers to the ability of host plantto produce a larger yield of good quality than other plants when beingfed upon by similar numbers of insects.

In a preferred aspect, the present invention provides a soybean plant tobe assayed for resistance or susceptibility to aphids by any method todetermine whether a soybean plant is very resistant, resistant,moderately resistant, moderately susceptible, or susceptible.

In this aspect, a plant is assayed for aphid resistance orsusceptibility by visually estimating the number of aphids on a plant(Mensah et al. Crop Sci 25:2228-2233 (2005)).

As used herein, aphid resistance refers to preventing or inhibiting theability of aphids to cause damage, such as reducing feeding, delayinggrowth and developing, reducing fecundity and the like, to a host plant.

In another aspect, the soybean plant can show a comparative resistancecompared to a non-resistant control soybean plant. In this aspect, acontrol soybean plant will preferably be genetically similar except forthe aphid resistant allele or alleles in question. Such plants can begrown under similar conditions with equivalent or near equivalentexposure to the pest. In this aspect, the resistant plant or plants hassignificantly fewer aphids per plant or damage per plant on resistantplants compared to known susceptible plants, and equivalent number ofaphids or damage per plant compared to known resistant plants

As used herein, the terms quantitative trait loci and QTL refer to agenomic region affecting the phenotypic variation in continuouslyvarying traits like yield or resistance. A QTL can comprise multiplegenes or other genetic factors even within a contiguous genomic regionor linkage group.

As used herein, the terms single nucleotide polymorphism and SNP referto a single base difference between two DNA sequences.

As used herein, the term oligonucleotide refers to a molecule comprisedof two or more deoxyribonucleotides or ribonucleotides.

As used herein, the term primer refers to an oligonucleotidecomplementary to a given nucleotide sequence and that is needed toinitiate replication by a polymerase.

As used herein, the term probe refers to an oligonucleotide that iscapable of hybridizing to another oligonucleotide of interest. A probemay be a single-stranded or double stranded oligonucleotide. Probes areuseful for detection, identification or isolation of particularnucleotide sequence.

As used herein, the term gene refers to a nucleic acid sequence thatcomprises introns, untranslated regions and control regions, and codingsequences necessary for the production RNA, a polypeptide or apre-cursor of a polypeptide.

As used herein, the term marker, DNA marker, and genetic marker refersto a trait, including genetic traits such as DNA sequences loci alleleschromosome features isozyme, and morphological traits that can be usedas detect the presence or location of a gene or trait in an individualor in a population.

As used herein, a diagnostic marker refers to a genetic marker than candetect or identify a trait, examples of which including aphidresistance, rust resistance and yield.

A resistance QTL of the present invention may be introduced into anelite soybean line. Herein, “line” refers to a group of individualplants from the similar parentage with similar traits. An “elite line”is any line that has resulted from breeding and selection for superioragronomic performance. Additionally, an elite line is sufficientlyhomogenous and homozygous to be used for commercial production. Elitelines may be used in the further breeding efforts to develop new elitelines.

An aphid resistance QTL of the present invention may also be introducedinto an soybean line comprising one or more transgenes conferringtransgenic plant that contains one or more genes for herbicidetolerance, increased yield, insect control, fungal disease resistance,virus resistance, nematode resistance, bacterial disease resistance,mycoplasma disease resistance, modified oils production, high oilproduction, high protein production, germination and seedling growthcontrol, enhanced animal and human nutrition, low raffinose,environmental stress resistant, increased digestibility, industrialenzymes, pharmaceutical proteins, peptides and small molecules, improvedprocessing traits, improved flavor, nitrogen fixation, hybrid seedproduction, reduced allergenicity, biopolymers, and biofuels amongothers. These agronomic traits can be provided by the methods of plantbiotechnology as transgenes in soybean.

An aphid resistant QTL allele or alleles can be introduced from anyplant that contains that allele (donor) to any recipient soybean plant.In one aspect, the recipient soybean plant can contain additional aphidresistant loci. In another aspect, the recipient soybean plant cancontain a transgene. In another aspect, while maintaining the introducedQTL, the genetic contribution of the plant providing the aphid resistantQTL can be reduced by back-crossing or other suitable approaches. In oneaspect, the nuclear genetic material derived from the donor material inthe soybean plant can be less than or about 50%, less than or about 25%,less than or about 13%, less than or about 5%, 3%, 2% or 1%, but thatgenetic material contains the aphid resistant locus or loci of interest.

Plants containing one or more aphid resistant loci described can bedonor plants. Aphid plants containing resistant loci can be, examples ofwhich including screened for by using a nucleic acid molecule capable ofdetecting a marker polymorphism associated with resistance. In oneaspect, a donor plant is PI 594427C. In a preferred aspect, a donorplant is the source for aphid resistance loci 1, 2, 4, 6, 7, 8, 9, 11,13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 27, and 28. In another aspect, adonor plant is soybean variety MV0031. In another preferred aspect, adonor plant is the source for aphid resistance loci 2, 5, 8, 10, 25, and26. In another aspect, a donor plant is soybean variety CNS (PI 548445).In another preferred aspect, a donor plant is the source for aphidresistance loci 3, 12, 14, and 24. A donor plant can be a susceptibleline. In one aspect, a donor plant can also be a recipient soybeanplant.

As used herein, a maturity group refers to an industry division ofgroups of varieties based range in latitude which the plant is bestadapted and most productive. Soybean varieties are classified into 13recognized maturity groups with the designations ranging from maturitygroups 000, 00, 0, and I through X, wherein 000 represents the earliestmaturing variety and X represents the latest maturing variety. Soybeanplants in maturity groups 000 to IV have indeterminate plant habit,while soybean plants in maturity groups V through X have determinateplant habit. Herein, determinate growth habit refers to a ceasevegetative growth after the main stem terminates in a cluster of maturepods. Herein, indeterminate growth habit refers to the development ofleaves and flowers simultaneously throughout a portion of theirreproductive period, with one to three pods at the terminal apex. Earlymaturity varieties (000 to IV) are adapted to northern latitudes withlonger day lengths with the maturity designation increasing in southernlatitudes with shorter day lengths

Herein, relative maturity refers to a soybean plant maturity groupsubdividing a maturity group into tenths, for example III.5. Relativematurity provided a more exact maturity. The number following thedecimal point refers to the relative earliness or lateness with amaturity group, examples of which including IV.2 is an early group IVvariety and IV.9 is a late group IV.

It is further understood that a soybean plant of the present inventionmay exhibit the characteristics of any relative maturity group. In anaspect, the relative maturity group is selected from the groupconsisting of 000.1-000.9, 00.1-00.9, 0.1-0.9, I.1-I.9, II.1-II.9,III.1-III.9, IV.1-IV.9, V.1-V.9, VI.1-VI.9, VII.1-VII.9, VIII.1-VIII.9,IX.1-IX.9, and X.1-X.9. The pollen for selected soybean plant can becryopreserved and used in crosses with soybean lines from other maturitygroups to introgress an aphid resistance locus in a line that would notnormally be available for crossing in nature. Pollen cryopreservationtechniques are well known in the art (Tyagi and Hymowitz, Cryo letters24: 119-124 (2003), Liang et al. Acta Botanica Sinica 35: 733-738(1993)).

The aphid resistance effect of the QTL can vary based on parentalgenotype and on the environmental factors in which the aphid resistanceis measured. It is within the skill of those in the art of plantbreeding and without undue experimentation to use methods describedherein to select from populations of plants or from a collection ofparental genotypes those that when containing an aphid resistance locusresult in enhanced aphid resistance relative to the parental genotype.Herein, an infestation can be caused by insects, fungi, virus, bacteriumor invertebrate animal.

A number of molecular genetic maps of Glycine have been reported (Mansuret al., Crop Sci. 36: 1327-1336 (1996), Shoemaker et al., Genetics 144:329-338 (1996); Shoemaker et al., Crop Science 32: 1091-1098 (1992),Shoemaker et al., Crop Science 35: 436-446 (1995); Tinley and Rafalski,J. Cell Biochem. Suppl. 14E: 291 (1990),); Cregan et al., Crop Science39:1464-1490 (1999). Glycine max, Glycine soja and Glycine max x.Glycine soja share linkage groups (Shoemaker et al., Genetics 144:329-338 (1996). A linkage group (LG) is a set of genes that tend to beinherited together from generation to generation. As used herein,reference to the linkage groups (LG), J, E, B1, N, A1, D1a_Q, H, D1, F,I D1b+W, O C1 and B2 of Glycine max refers to the linkage group thatcorresponds to linkage groups, J, E, B1, N, A1, D1a_Q, H, D1, F, ID1b+W, O C1 and B2 from the genetic map of Glycine max (Mansur et al.,Crop Science 36: 1327-1336 (1996); Cregan et al., Crop Science39:1464-1490 (1999), and Soybase, Agricultural Research Service, UnitedStates Department of Agriculture).

An allele of a QTL can, of course, comprise multiple genes or othergenetic factors even within a contiguous genomic region or linkagegroup, such as a haplotype. A linkage group is a group loci carried onthe same chromosome. A haplotype is set of genetic markers associatedwith closely linked segments of DNA on one chromosome and tend to beinherited as a unit. As used herein, an allele of a resistance locus cantherefore encompass more than one gene or other genetic factor whereineach individual gene or genetic component is also capable of exhibitingallelic variation and wherein each gene or genetic factor is alsocapable of eliciting a phenotypic effect on the quantitative trait inquestion. In an aspect of the present invention the allele of a QTLcomprises one or more genes or other genetic factors that are alsocapable of exhibiting allelic variation. The use of the term “an alleleof a QTL” is thus not intended to exclude a QTL that comprises more thanone gene or other genetic factor. Specifically, an “allele of a QTL” inthe present invention can denote a haplotype within a haplotype windowwherein a phenotype can be pest resistance. A haplotype window is acontiguous genomic region that can be defined, and tracked, with a setof one or more polymorphic markers wherein the polymorphisms indicateidentity by descent. A haplotype within that window can be defined bythe unique fingerprint of alleles at each marker. As used herein, anallele is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When all the alleles present at a given locus ona chromosome are the same, that plant is homozygous at that locus. Ifthe alleles present at a given locus on a chromosome differ, that plantis heterozygous at that locus. Plants of the present invention may behomozygous or heterozygous at any particular aphid resistance locus orfor a particular polymorphic marker.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred aspect of thepresent invention, the plant part is a seed.

Plants or parts thereof of the present invention may be grown in cultureand regenerated. Methods for the regeneration of Glycine max plants fromvarious tissue types and methods for the tissue culture of Glycine maxare known in the art (See, examples of which including Widholm et al.,In Vitro Selection and Culture-induced Variation in Soybean, In Soybean:Genetics, Molecular Biology and Biotechnology, Eds. Verma and Shoemaker,CAB International, Wallingford, Oxon, England (1996). Regenerationtechniques for plants such as Glycine max can use as the startingmaterial a variety of tissue or cell types. With Glycine max inparticular, regeneration processes have been developed that begin withcertain differentiated tissue types such as meristems, Cartha et al.,Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections, Cameya et al.,Plant Science Letters 21: 289-294 (1981), and stem node segments, Sakaet al., Plant Science Letters, 19: 193-201 (1980); Cheng et al., PlantScience Letters, 19: 91-99 (1980). Regeneration of whole sexually matureGlycine max plants from somatic embryos generated from explants ofimmature Glycine max embryos has been reported (Ranch et al., In VitroCellular & Developmental Biology 21: 653-658 (1985). Regeneration ofmature Glycine max plants from tissue culture by organogenesis andembryogenesis has also been reported (Barwale et al., Planta 167:473-481 (1986); Wright et al., Plant Cell Reports 5: 150-154 (1986).

The present invention also provides an aphid resistant soybean plantselected for by screening for aphid resistance or susceptibility in thesoybean plant, the selection comprising interrogating genomic nucleicacids for the presence of a marker molecule that is genetically linkedto an allele of a QTL associated with aphid resistance in the soybeanplant, where the allele of a QTL is also located on a linkage groupassociated with aphid resistant soybean.

The present invention includes a method of introgressing an aphidresistant allele into a soybean plant comprising (A) crossing at leastone first soybean plant comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 81 through SEQ ID NO: 120 with atleast one second soybean plant in order to form a segregatingpopulation, (B) screening the segregating population with one or morenucleic acid markers to determine if one or more soybean plants from thesegregating population contains the nucleic acid sequence, and (C)selecting from the segregation population one or more soybean plantscomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 81 through SEQ ID NO: 120.

The present invention includes a method of introgressing an allele intoa soybean plant comprising: (A) crossing at least one aphid resistantsoybean plant with at least one aphid sensitive soybean plant in orderto form a segregating population; (B) screening said segregatingpopulation with one or more nucleic acid markers to determine if one ormore soybean plants from said segregating population contains an aphidresistant allele, wherein said aphid resistance allele is an alleleselected from the group consisting of aphid resistance allele 1, aphidresistance allele 2, aphid resistance allele 3, aphid resistance allele4, aphid resistance allele 5, aphid resistance allele 6, aphidresistance allele 7, aphid resistance allele 8, aphid resistance allele9, aphid resistance allele 10, aphid resistance allele 11, aphidresistance allele 12, aphid resistance allele 13, aphid resistanceallele 14, aphid resistance allele 15, aphid resistance allele 16, aphidresistance allele 17, aphid resistance allele 18, aphid resistanceallele 19, aphid resistance allele 20, aphid resistance allele 21, aphidresistance allele 22, aphid resistance allele 23, aphid resistanceallele 24, aphid resistance allele 25, aphid resistance allele 26, aphidresistance allele 27, aphid resistance allele 28, aphid resistanceallele 29, aphid resistance allele 30, aphid resistance allele 31, aphidresistance allele 32, aphid resistance allele 33, aphid resistanceallele 34, aphid resistance allele 35, aphid resistance allele 36, aphidresistance allele 37, aphid resistance allele 38, aphid resistanceallele 39, and aphid resistance allele 40.

The present invention includes nucleic acid molecules. Such moleculesinclude those nucleic acid molecules capable of detecting a polymorphismgenetically or physically linked to aphid resistance loci. Suchmolecules can be referred to as markers. Additional markers can beobtained that are linked to aphid resistance locus 1, aphid resistancelocus 2, aphid resistance locus 3, aphid resistance locus 4, aphidresistance locus 5, aphid resistance locus 6, aphid resistance locus 7,aphid resistance locus 8, aphid resistance locus 9, aphid resistancelocus 10, aphid resistance locus 11, aphid resistance locus 12, aphidresistance locus 13, aphid resistance locus 14, aphid resistance locus15, aphid resistance locus 16, aphid resistance locus 17, aphidresistance locus 18, aphid resistance locus 19, aphid resistance locus20, aphid resistance locus 21, aphid resistance locus 22, aphidresistance locus 23, aphid resistance locus 24, aphid resistance locus25, aphid resistance locus 26, aphid resistance locus 27, and aphidresistance locus 28 by available techniques. In one aspect, the nucleicacid molecule is capable of detecting the presence or absence of amarker located less than 50, 40, 30, 20, 10, 5, 2, or 1 centimorgansfrom an aphid resistance loci. In another aspect, a marker exhibits aLOD score of 2 or greater, 3 or greater, or 4 or greater with aphidresistance, measuring using MapManager or QGene Version 3 and defaultparameters. In another aspect, the nucleic acid molecule is capable ofdetecting a marker in a locus selected from the group aphid resistancelocus 1, aphid resistance locus 2, aphid resistance locus 3, aphidresistance locus 4, aphid resistance locus 5, aphid resistance locus 6,aphid resistance locus 7, aphid resistance locus 8, aphid resistancelocus 9, aphid resistance locus 10, aphid resistance locus 11, aphidresistance locus 12, aphid resistance locus 13, aphid resistance locus14, aphid resistance locus 15, aphid resistance locus 16, aphidresistance locus 17, aphid resistance locus 18, aphid resistance locus19, aphid resistance locus 20, aphid resistance locus 21, aphidresistance locus 22, aphid resistance locus 23, aphid resistance locus24, aphid resistance locus 25, aphid resistance locus 26, aphidresistance locus 27, and aphid resistance locus 28. In a further aspect,a nucleic acid molecule is selected from the group consisting of SEQ IDNO: 81 through SEQ ID NO: 120, fragments thereof, complements thereof,and nucleic acid molecules capable of specifically hybridizing to one ormore of these nucleic acid molecules.

In a preferred aspect, a nucleic acid molecule of the present inventionincludes those that will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO: 81 through SEQ ID NO: 120or complements thereof or fragments of either under moderately stringentconditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred aspect, a nucleic acid of the present inventionwill specifically hybridize to one or more of the nucleic acid moleculesset forth in SEQ ID NO: 81 through SEQ ID NO: 120 or complements orfragments of either under high stringency conditions. In one aspect ofthe present invention, a preferred marker nucleic acid molecule of thepresent invention has the nucleic acid sequence set forth in SEQ ID NO:81 through SEQ ID NO: 120 or complements thereof or fragments of either.In another aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 80% and 100% or90% and 100% sequence identity with the nucleic acid sequence set forthin SEQ ID NO: 81 through SEQ ID NO: 120 or complement thereof orfragments of either. In a further aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 95% and 100% sequence identity with the sequence set forth inSEQ ID NO: 81 through SEQ ID NO: 120 or complement thereof or fragmentsof either. In a more preferred aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 98% and 100% sequence identity with the nucleic acid sequenceset forth in SEQ ID NO: 81 through SEQ ID NO: 120 or complement thereofor fragments of either.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules are exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12: 203-213; andWetmur et al. 1968 J. Mol. Biol. 31:349-370. Appropriate stringencyconditions that promote DNA hybridization are, examples of whichincluding 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y., 1989, 6.3.1-6.3.6. Examples of which including thesalt concentration in the wash step can be selected from a lowstringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed.

Examples of which including hybridization using DNA or RNA probes orprimers can be performed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100μg/mL nonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.

A fragment of a nucleic acid molecule can be any sized fragment andillustrative fragments include fragments of nucleic acid sequences setforth in SEQ ID NO: 81 through SEQ ID NO: 120 and complements thereof.In one aspect, a fragment can be between 15 and 25, 15 and 30, 15 and40, 15 and 50, 15 and 100, 20 and 25, 20 and 30, 20 and 40, 20 and 50,20 and 100, 25 and 30, 25 and 40, 25 and 50, 25 and 100, 30 and 40, 30and 50, and 30 and 100. In another aspect, the fragment can be greaterthan 10, 15, 20, 25, 30, 35, 40, 50, 100, or 250 nucleotides.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with aphid resistance of soybean of the presentinvention. Examples of public marker databases include, for example:Soybase, Agricultural Research Service, United States Department ofAgriculture.

A genetic marker is a DNA sequence that has a known location on achromosome and associated with a particular trait or gene. Geneticmarkers associated with aphid resistance can be used to determinewhether an individual plant is resistant to aphids.

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations wherein individuals are predominantly homozygous and lociare predominantly dimorphic, dominant and codominant markers can beequally valuable. As populations become more heterozygous andmultiallelic, codominant markers often become more informative of thegenotype than dominant markers.

Markers, such as single sequence repeat markers (SSR), AFLP markers,RFLP markers, RAPD markers, phenotypic markers, SNPs, isozyme markers,microarray transcription profiles that are genetically linked to orcorrelated with alleles of a QTL of the present invention can beutilized (Walton, 1993; Burow et al. 1988). Methods to isolate suchmarkers are known in the art.

The detection of polymorphic sites in a sample of DNA, RNA, or cDNA maybe facilitated through the use of nucleic acid amplification methods.Such methods specifically increase the concentration of polynucleotidesthat span the polymorphic site, or include that site and sequenceslocated either distal or proximal to it. Such amplified molecules can bereadily detected by gel electrophoresis, fluorescence detection methods,or other means.

A method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent No. 50,424; European Patent No. 84,796;European Patent No. 258,017; European Patent No. 237,362; EuropeanPatent No. 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and 4,683,194),using primer pairs that are capable of hybridizing to the proximalsequences that define a polymorphism in its double-stranded form.

For the purpose of QTL mapping, the markers included should bediagnostic of origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is very low. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander et al. (Lander et al. 1989 Genetics,121:185-199), and the interval mapping, based on maximum likelihoodmethods described therein, and implemented in the software packageMAPMAKER/QTL (Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990). Additional software includes Qgene, Version 3,Department of Plant Breeding and Biometry, 266 Emerson Hall, CornellUniversity, Ithaca, N.Y.). Use of Qgene software is a particularlypreferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀(MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander etal. (1989), and further described by Arús and Moreno-González, PlantBreeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak et al. 1995 Genetics, 139:1421-1428).Multiple regression methods or models can be also be used, in which thetrait is regressed on a large number of markers (Jansen, Biometrics inPlant Breed, van Oijen, Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 116-124 (1994); Weber and Wricke, Advances in Plant Breeding,Blackwell, Berlin, 16 (1994)). Procedures combining interval mappingwith regression analysis, whereby the phenotype is regressed onto asingle putative QTL at a given marker interval, and at the same timeonto a number of markers that serve as ‘cofactors,’ have been reportedby Jansen et al. (Jansen et al. 1994 Genetics, 136:1447-1455) and Zeng(Zeng 1994 Genetics 136: 1457-1468). Generally, the use of cofactorsreduces the bias and sampling error of the estimated QTL positions (Utzand Melchinger, Biometrics in Plant Breeding, van Oijen, Jansen (eds.)Proceedings of the Ninth Meeting of the Eucarpia Section Biometrics inPlant Breeding, The Netherlands, pp. 195-204 (1994), thereby improvingthe precision and efficiency of QTL mapping (Zeng 1994). These modelscan be extended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al. 1995 Theor. Appl.Genet. 91:33-3).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingin plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (eds.). Plenum Press, New York,pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

An F₂ population is the first generation of selfing after the hybridseed is produced. Usually a single F₁ plant is selfed to generate apopulation segregating for all the genes in Mendelian (1:2:1) fashion.Maximum genetic information is obtained from a completely classified F₂population using a codominant marker system (Mather, Measurement ofLinkage in Heredity: Methuen and Co., (1938)). In the case of dominantmarkers, progeny tests (e.g. F₃, BCF₂) are required to identify theheterozygotes, thus making it equivalent to a completely classified F₂population. However, this procedure is often prohibitive because of thecost and time involved in progeny testing. Progeny testing of F₂individuals is often used in map construction where phenotypes do notconsistently reflect genotype (e.g. pest resistance) or where traitexpression is controlled by a QTL. Segregation data from progeny testpopulations (e.g. F₃ or BCF₂) can be used in map construction.Marker-assisted selection can then be applied to cross progeny based onmarker-trait map associations (F₂, F₃), where linkage groups have notbeen completely disassociated by recombination events (i.e., maximumdisequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al. 1992 Proc. Natl. Acad. Sci.(USA) 89:1477-1481). However, as the distance between markers becomeslarger (i.e., loci become more independent), the information in RILpopulations decreases dramatically.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdcomprising of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al. 1992). Information obtained from backcross populations usingeither codominant or dominant markers is less than that obtained from F₂populations because one, rather than two, recombinant gametes aresampled per plant. Backcross populations, however, are more informative(at low marker saturation) when compared to RILs as the distance betweenlinked loci increases in RIL populations (i.e. about 0.15%recombination). Increased recombination can be beneficial for resolutionof tight linkages, but may be undesirable in the construction of mapswith low marker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant-analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al. 1991 Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular pests) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.2006 Genetics 172:663-686). This approach tracks blocks of DNA known ashaplotypes, as defined by polymorphic markers, which are assumed to beidentical by descent in the mapping population. This assumption resultsin a larger effective sample size, offering greater resolution of QTL.Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). A cultivar is a race or variety of a plantspecies that has been created or selected intentionally and maintainedthrough cultivation.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) on the progeny of any cross. It isunderstood that nucleic acid markers of the present invention can beused in a MAS (breeding) program. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors such as, examples of which including emergence vigor,vegetative vigor, stress tolerance, disease resistance, branching,flowering, seed set, seed size, seed density, standability, andthreshability etc. will generally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredaspect, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease and insect-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

The development of new elite soybean hybrids requires the developmentand selection of elite inbred lines, the crossing of these lines andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Additional data on parental lines, as well as thephenotype of the hybrid, influence the breeder's decision whether tocontinue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have most attributes of the recurrentparent (e.g., cultivar) and, in addition, the desirable traittransferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of cropimprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant breeding perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979; Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Manograph., 16:249, 1987; Fehr,“Principles of variety development,” Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376, 1987).

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising thenucleic acid molecules of the present invention.

As used herein, a “nucleic acid molecule,” be it a naturally occurringmolecule or otherwise may be “substantially purified”, if desired,referring to a molecule separated from substantially all other moleculesnormally associated with it in its native state. More preferably asubstantially purified molecule is the predominant species present in apreparation. A substantially purified molecule may be greater than 60%free, preferably 75% free, more preferably 90% free, and most preferably95% free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “substantially purified” is not intended toencompass molecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal. 1987 Science 238:336-340; Albarella et al., European Patent No.144914), chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi etal., European Patent No. 119448).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1 Identifying Antixenosis Type Aphid Resistance inPI594427C

Soybean breeders evaluate soybean gerrnplasm for genetic traits thatconfer resistance to attack and injury by aphids. Host plant resistanceis classified as antixenosis, antibiosis or tolerance. Antixenosis, alsoreferred to as non-preference, is the ability of a plant to repelinsects, causing a reduction in oviposition or feeding. Dowling, CNS (PI548445). Jackson, PI 597727C, PI 594403 and Williams were evaluated forantixenosis type resistance. Antixenosis resistance is typical evaluatedchoice experiments where insects can select between at least two hostplants.

PI 594427C was identified to have resistance to aphids in choice fieldtests conducted at Michigan State University over three field season. Inthe choice field tests, Dowling, CNS, Jackson, PI 597727C, PI 594403 andWilliams were grown and enclosed in a single cage. Two field collectedaphids were placed on each plant. Aphids were able to move freely fromplant to plant, therefore the study evaluated the aphid plantpreference. Plants were individually scored at 2, 3, 4, or 5 wks afterinfestation, depending on when symptoms were first observed. Plants weregiven a visual rating ranging from 0 to 4 (Table 1).

TABLE 1 Description of rating scale used for aphid resistancephenotyping Rating Symptoms 0 Very Resistant No aphids 1 Resistant Fewerthan 100 aphids 2 Moderately Resistant 101-300 aphids 3 ModeratelySusceptible 300-800 aphids 4 Susceptible >800 aphidsEach week, the plants were also assigned a damage index (DI), which iscalculated using the following formula:

${DI} = {\frac{\Sigma( {{each}\mspace{14mu}{scale} \times {{no}.\mspace{11mu}{of}}\mspace{11mu}{plants}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{scale}} )}{4 \times {total}\mspace{14mu}{{no}.\mspace{11mu}{of}}\mspace{11mu}{plants}\mspace{14mu}{evaluated}} \times 100}$A higher damage index corresponds to a more susceptible plant.

Over three years of field tests, PI 594427C consistently showedequivalent or lower aphid ratings and damage indices than known aphidresistant varieties, Jackson and Dowling (Table 2). CNS and Williamswere sensitive to aphid infestation.

TABLE 2 Average soybean aphid rating and damage index foraphid-resistant and -susceptible soybean genotypes tested in triplicatein field cages over 3 years at Michigan State University. Aphid ratingDamage index^(¶) Line (0-4) (%) Dowling 1.8 44 CNS 3.8 95 Jackson 1.9 48PI 594427C 1.5 37 PI 594403 2.5 63 Williams 3.8 94

Example 2 Identifying Antibiosis Type Aphid Resistance in PI1594427C

Host plant resistance is classified as antixenosis, antibiosis ortolerance. Antibiosis is the ability of a plant to reduce the survival,growth, or reproduction of insects that feed on it. Antibiosis is oftencaused by the production of toxic chemicals by the plant. Antibiosistype plant resistance is often evaluated in no-choice studies whereinsect are supplied a single food source. Pana, PI 594403, PI 71506,Williams, PI594403, PI 594427C, CNS, Jackson, and Dowling were evaluatedfor antibiosis resistance in no choice experiments.

Antibiosis resistance was identified in PI 594427C in no choiceconducted at University of Illinois. Plants were grown in isolated cagesin a choice situation. Three aphid nymphs were placed on each plant. Thenumber of aphids was counted at 14, 17, and 21 days after inoculation(Table 2). Four replications were performed for each entry. Aphids wereable to readily reproduce on CNS under antixenosis conditions (Table 2),but not under antibiosis conditions. The level of aphid infestation washigher and the aphid age range was broader under the antixenosisevaluation compared to aphid infestation and age range under theantibiosis evaluation. The difference in aphid infestation level andaphid maturity may account for the differences observed on CNS in theantibiosis and antixenosis evaluations. Furthermore, geographic aphidbiotypes from Illinois and Michigan may account for differences inreaction on CNS.

TABLE 3 Average number of soybean aphid for aphid-resistant and aphid-susceptible soybean genotypes tested at University of Illinois. No. ofAphids per Plant at: Line 14 d 17 d 21 d* Pana 190 405 1076a   PI 594403142 497 603a  PI 71506 200 316 294ab  Williams 126 251 236ab  PI 59440336 83 59bc PI 594427C 20 17 12cd CNS 21 22 11cd CNS 17 24 10cd Jackson14 5 6d PI 594427C 17 12 5d Dowling 7 3 3d Dowling 6 3 3d *Meansfollowed by different letters are significantly different at 0.05 level.

Example 3 Aphid Mapping Studies

In order to map putative QTL to aphid resistance, a resistant line (PI594427C) was crossed with a CNS or MV0031. Two mapping populations weredeveloped to map putative QTLs linked with aphid resistance: PI594427C(aphid resistant)×MV0031 and PI594427C (aphid resistant)×CNS. F₃:PI594427C×MV0031 and F₄: PI594427C×CNS populations were evaluated foraphid resistance phenotype in enclosed cages in East Lansing, Mich.Three aphid nymphs were placed per plant and aphid density was rated at3, 4, 5 weeks after inoculation. The rating scale was 0-5 (Table 1).Twenty-eight aphid resistant loci were identified (Table 6 and 7).

The phenotype data from week 4 evaluation was used from QTL mappingstudies (Tables 4 and 5). At week 4, the aphid resistant parent,PI594427C, was rated 1.5 and the aphid sensitive parents, MV0031 andCNS, were rated 3.5 In addition to the above-described phenotyping, eachpopulation was genotyped with SNP markers: 181 polymorphic SNPs with theF₃: PI594427C×MV0031 population and 164 polymorphic SNPs with the F₄:PI594427C×CNS population. Single marker and marker regression analyseswere performed to determine QTL regions conditioning aphid resistance.Tables 6 and 7 list significant associations between genomic regions andaphid resistance along with diagnostic markers.

TABLE 4 Phenotype of F₄: PI594427CxCNS at week 4 after inoculation.Aphid Rating Number of Plants 0.5 0 1 4 1.5 8 2 32 2.5 64 3 70 3.5 6 4 0Total (n) 184

TABLE 5 Phenotype of F₃: PI594427CxMV0031 at week 4 after inoculation.Aphid Rating Number of Plants 0.5 0 1 2 1.5 5 2 29 2.5 29 3 88 3.5 30 40 Total (n) 183

TABLE 6 Results of markers associated with aphid resistance determined(ns = not significant) Aphid Resistance p- R-sq. Marker Population LociLG Marker Allele t-value* value** value*** Interval PI 594427C x CNS 1 JNS0115450 T 3.36(P < 0.05) 0.00118 8 38-58 cM PI 594427C x MV0031 1 JNS0122151 A 5.51(P < 0.05) <0.0001 14 28-48 cM PI 594427C x MV0031 2 ENS0126797 G 2.49(P < 0.05) 0.04961 3 30-50 cM PI 594427C x CNS 2 ENS0126797 A 2.01(P < 0.05) ns 3 30-50 cM PI 594427C x CNS 3 E NS0098210C 2.78(P < 0.05) 0.00741 5 76-96 cM PI 594427C x CNS 4 E NS0099483 C2.64(P < 0.05) 0.01348 5 111-131 cM PI 594427C x MV0031 5 B1 NS0100200 A2.04(P < 0.05) ns 2 44-64 cM PI 594427C x CNS 6 N NS0137720 C ns 0.029784  0-10 cM PI 594427C x CNS 7 G NS0118422 T ns 0.02406 4 77-97 cM PI594427C x MV0031 8 N NS0125467 T 2.03(P < 0.05) ns 3 26-46 cM PI 594427Cx CNS 8 N NS0129030 C ns 0.04662 4 15-35 cM PI 594427C x CNS 9 NNS0098575 T 2.03(P < 0.05) 0.04894 3 97-117 cM PI 594427C x MV0031 10 A1NS0129617 C 2.06(P < 0.05) 0.04753 4  0-15 cM PI 594427C x MV0031 11 A1NS0130304 A  2.2(P < 0.05) ns 3 33-53 cM PI 594427C x CNS 12 D1aNS0095317 I 2.51(P < 0.05) 0.00628 6 12-32 cM PI 594427C x CNS 13 C2NS0115731 A ns 0.02083 4 54-74 cM PI 594427C x CNS 14 H NS0120346 T2.28(P < 0.05) ns 3  1-13 cM PI 594427C x CNS 15 H NS0097165 A 2.03(P <0.05) ns 3 62-82 cM PI 594427C x MV0031 16 D2 NS0092748 T 4.44(P < 0.05)0.00008 10  0-18 cM PI 594427C x MV0031 16 D2 NS0096662 4.44 0.00008 0-18 cM PI 594427C x CNS 16 D2 NS0118525 T 2.37(P < 0.05) ns 3  0-10 cMPI 594427C x MV0031 17 F NS0099503 T 3.21(P < 0.05) 0.0049 6  0-18 cM PI594427C x CNS 18 F NS0123719 A 2.97(P < 0.05) 0.0062 6 62-82 cM PI594427C x MV0031 19 I NS0130766 A 2.47(P < 0.05) 0.02812 4  0-14 cM PI594427C x CNS 20 D1b NS0121903 C 3.23(P < 0.05) 0.00633 6 30-50 cM PI594427C x CNS 21 D1b NS0098438 A 2.01(P < 0.05) ns 2 124-144 cM PI594427C x CNS 21 D1b NS0114263 C ns 0.04948 3  91-111 cM PI 594427C xMV0031 22 O NS0124919 C 2.06(P < 0.05) ns 4 50-70 cM PI 594427C x CNS 23O NS0124051 C 2.72(P < 0.05) 0.00485 6 120-140 cM PI 594427C x CNS 24 C1NS0124300 G 2.64(P < 0.05) 0.02273 4  0-15 cM PI 594427C x MV0031 25 C1NS0093331 C 2.04(P < 0.05) ns 3 28-48 cM PI 594427C x MV0031 26 C1NS0097882 T 2.02(P < 0.05) ns 3 113-133 cM PI 594427C x MV0031 26 C1NS0136956 G ns 0.00561 6 72-92 cM PI 594427C x CNS 27 K NS0098803 T ns0.02604 4  0-20 cM PI 594427C x MV0031 28 B2 NS0092589 G 2.21(P < 0.05)0.03209 5  7-27 cM PI 594427C x CNS 28 B2 NS0103077 D 2.18(P < 0.05)0.0415 4  0-10 cM *Marker analysis was performed using a t-test. P-value≦ 0.05 is significant. **Marker analysis was performed using markerregression in MapManager QTX; ***R-squared value from marker regressionin MapManager QTX

TABLE 7 Listing of SNP markers for aphid resistance loci 1-28 Aphid SEQSEQ Resis- Resis- SEQ ID SEQ ID ID ID tance tance SEQ Forward ReverseVIC FAM Loci Marker Allele ID Primer Primer probe probe 1 NS0115450 T 811 2 121 122 1 NS0122151 A 82 3 4 123 124 1 NS0125096 83 5 6 125 126 1NS0120948 84 7 8 127 128 2 NS0126797 G 85 9 10 129 130 3 NS0098210 C 8611 12 131 132 4 NS0099483 C 87 13 14 133 134 5 NS0100200 A 88 15 16 135136 6 NS0137720 C 89 17 18 137 138 7 NS0118422 T 90 19 20 139 140 8NS0125467 T 91 21 22 141 142 8 NS0129030 C 92 23 24 143 144 9 NS0098575T 93 25 26 145 146 10 NS0129617 C 94 27 28 147 148 11 NS0130304 A 95 2930 149 150 12 NS0095317 I 96 31 32 151 152 13 NS0115731 A 97 33 34 153154 14 NS0120346 T 98 35 36 155 156 15 NS0097165 A 99 37 38 157 158 16NS0092748 T 100 39 40 159 160 16 NS0096662 101 41 42 161 162 16NS0118525 T 102 43 44 163 164 17 NS0099503 T 103 45 46 165 166 18NS0123719 A 104 47 48 167 168 19 NS0130766 A 105 49 50 169 170 20NS0121903 C 106 51 52 171 172 21 NS0098438 A 107 53 54 173 174 21NS0114263 C 108 55 56 175 176 22 NS0124919 C 109 57 58 177 178 23NS0124051 C 110 59 60 179 180 23 NS0118907 111 61 62 181 182 24NS0124300 G 112 63 64 183 184 25 NS0093331 C 113 65 66 185 186 26NS0097882 T 114 67 68 187 188 27 NS0098803 T 116 71 72 191 192 28NS0092589 G 117 73 74 193 194 28 NS0103077 D 118 75 76 195 196 28NS0100457 119 77 78 197 198 28 NS0116259 120 79 80 199 200

SNP markers determined to be associated with region 1 are SEQ ID NO: 81through SEQ ID NO: 84. SNP markers for region 1 are mapped to a regionon linkage group J. Table 7 lists sequences for PCR amplificationprimers, indicated as SEQ ID NO: 1 through SEQ ID NO: 8 and probesindicated as SEQ ID NO: 121 through SEQ ID NO: 128.

A SNP marker determined to be associated with region 2 is SEQ ID NO: 85.A SNP marker for region 2 is mapped to a region on linkage group E.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 9 through SEQ ID NO: 10 and probes indicated as SEQ ID NO: 129through SEQ ID NO: 130.

A SNP marker determined to be associated with region 3 is SEQ ID NO: 86.A SNP marker for region 3 is mapped to a region on linkage group E.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 11 through SEQ ID NO: 12 and probes indicated as SEQ ID NO: 131through SEQ ID NO: 132.

A SNP marker determined to be associated with region 4 is SEQ ID NO: 87.A SNP marker for region 4 is mapped to a region on linkage group E.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 13 through SEQ ID NO: 14 and probes indicated as SEQ ID NO: 133through SEQ ID NO: 134.

A SNP marker determined to be associated with region 5 is SEQ ID NO: 88.A SNP marker for region 5 is mapped to a region on linkage group B1.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 15 through SEQ ID NO: 16 and probes indicated as SEQ ID NO: 135through SEQ ID NO: 136.

A SNP marker determined to be associated with region 6 is SEQ ID NO: 89.A SNP marker for region 6 is mapped to a region on linkage group N.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 17 through SEQ ID NO: 18 and probes indicated as SEQ ID NO: 137through SEQ ID NO: 138.

A SNP marker determined to be associated with region 7 is SEQ ID NO: 90.A SNP marker for region 7 is mapped to a region on linkage group G.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 19 through SEQ ID NO: 20 and probes indicated as SEQ ID NO: 139through SEQ ID NO: 140.

SNP markers determined to be associated with region 8 are SEQ ID NO: 91through SEQ ID NO: 92. SNP markers for region 8 are mapped to a regionon linkage group N. Table 7 lists sequences for PCR amplificationprimers, indicated as SEQ ID NO: 21 through SEQ ID NO: 24 and probesindicated as SEQ ID NO: 141 through SEQ ID NO: 144.

A SNP marker determined to be associated with region 9 is SEQ ID NO: 93.A SNP marker for region 9 is mapped to a region on linkage group N.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 25 through SEQ ID NO: 26 and probes indicated as SEQ ID NO: 145through SEQ ID NO: 146.

A SNP marker determined to be associated with region 10 is SEQ ID NO:94. A SNP marker for region 10 is mapped to a region on linkage groupA1. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 27 through SEQ ID NO: 28 and probes indicated as SEQ ID NO:147 through SEQ ID NO: 148.

A SNP marker determined to be associated with region 11 is SEQ ID NO:95. A SNP marker for region 11 is mapped to a region on linkage groupA1. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 29 through SEQ ID NO: 30 and probes indicated as SEQ ID NO:149 through SEQ ID NO: 150.

A SNP marker determined to be associated with region 12 is SEQ ID NO:96. A SNP marker for region 12 is mapped to a region on linkage groupD1a. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 31 through SEQ ID NO: 32 and probes indicated as SEQ ID NO:151 through SEQ ID NO: 152.

A SNP marker determined to be associated with region 13 is SEQ ID NO:97. A SNP marker for region 13 is mapped to a region on linkage groupC2. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 33 through SEQ ID NO: 34 and probes indicated as SEQ ID NO:153 through SEQ ID NO: 154.

A SNP marker determined to be associated with region 14 is SEQ ID NO:98. A SNP marker for region 14 is mapped to a region on linkage group H.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 35 through SEQ ID NO: 36 and probes indicated as SEQ ID NO: 155through SEQ ID NO: 156.

A SNP marker determined to be associated with region 15 is SEQ ID NO:99. A SNP marker for region 15 is mapped to a region on linkage group H.Table 7 lists sequences for PCR amplification primers, indicated as SEQID NO: 37 through SEQ ID NO: 38 and probes indicated as SEQ ID NO: 157through SEQ ID NO: 158.

SNP markers determined to be associated with region 16 are SEQ ID NO:100 through SEQ ID NO: 102. SNP markers for region 16 are mapped to aregion on linkage group D2. Table 7 lists sequences for PCRamplification primers, indicated as SEQ ID NO: 39 through SEQ ID NO: 42and probes indicated as SEQ ID NO: 159 through SEQ ID NO: 164.

A SNP marker determined to be associated with region 17 is SEQ ID NO:103. A SNP marker for region 17 is mapped to a region on linkage groupF. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 45 through SEQ ID NO: 46 and probes indicated as SEQ ID NO:165 through SEQ ID) NO: 166.

A SNP marker determined to be associated with region 18 is SEQ ID NO:104. A SNP marker for region 18 is mapped to a region on linkage groupF. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 47 through SEQ ID NO: 48 and probes indicated as SEQ ID NO:167 through SEQ ID NO: 168.

A SNP marker determined to be associated with region 19 is SEQ ID NO:105. A SNP marker for region 19 is mapped to a region on linkage groupI. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 49 through SEQ ID NO: 50 and probes indicated as SEQ ID NO:169 through SEQ ID NO: 170.

A SNP marker determined to be associated with region 20 is SEQ ID NO:106. A SNP marker for region 20 is mapped to a region on linkage groupD1b. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 51 through SEQ ID NO: 52 and probes indicated as SEQ ID NO:171 through SEQ ID NO: 172.

SNP markers determined to be associated with region 21 are SEQ ID NO:107 through SEQ ID NO: 108. SNP markers for region 21 are mapped to aregion on linkage group D1b. Table 7 lists sequences for PCRamplification primers, indicated as SEQ ID NO: 53 through SEQ ID NO: 56and probes indicated as SEQ ID NO: 173 through SEQ ID NO: 176.

A SNP marker determined to be associated with region 22 is SEQ ID NO:109. A SNP marker for region 22 is mapped to a region on linkage groupO. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 57 through SEQ ID NO: 58 and probes indicated as SEQ ID NO:177 through SEQ ID NO: 178.

SNP markers determined to be associated with region 23 are SEQ ID NO:110 SEQ ID NO: 11. A SNP marker for region 23 is mapped to a region onlinkage group O. Table 7 lists sequences for PCR amplification primers,indicated as SEQ ID NO: 59 through SEQ ID NO: 62 and probes indicated asSEQ ID NO: 179 through SEQ ID NO: 180.

A SNP marker determined to be associated with region 23 is SEQ ID NO:111. A SNP marker for region 23 is mapped to a region on linkage groupC1. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 61 through SEQ ID NO: 62 and probes indicated as SEQ ID NO:181 through SEQ ID NO: 182.

A SNP marker determined to be associated with region 24 is SEQ ID NO:112. A SNP marker for region 24 is mapped to a region on linkage groupC1. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 63 through SEQ ID NO: 64 and probes indicated as SEQ ID NO:183 through SEQ ID NO: 184.

A SNP marker determined to be associated with region 25 is SEQ ID NO:113. A SNP marker for region 25 is mapped to a region on linkage groupC1. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 65 through SEQ ID NO: 66 and probes indicated as SEQ ID NO:185 through SEQ ID NO: 186.

SNP markers determined to be associated with region 26 are SEQ ID NO:114 through SEQ ID NO: 116. SNP markers for region 26 are mapped to aregion on linkage group C1. Table 7 lists sequences for PCRamplification primers, indicated as SEQ ID NO: 67 through SEQ ID NO: 70and probes indicated as SEQ ID NO: 187 through SEQ ID NO: 190.

A SNP marker determined to be associated with region 27 is SEQ ID NO:116. A SNP marker for region 27 is mapped to a region on linkage groupK. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 71 through SEQ ID NO: 72 and probes indicated as SEQ ID NO:191 through SEQ ID NO: 192.

A SNP marker determined to be associated with region 28 is SEQ ID NO:117. SNP markers for region 28 are mapped to a region on linkage groupB2. Table 7 lists sequences for PCR amplification primers, indicated asSEQ ID NO: 73 through SEQ ID NO: 80 and probes indicated as SEQ ID NO:193 through SEQ ID NO: 200.

Example 4 Oligonucleotide Hybridization Probes Useful for DetectingSoybean Plants with Aphid Resistance Loci

Oligonucleotides can also be used to detect or type the polymorphismsassociated with aphid resistance disclosed herein by hybridization-basedSNP detection methods. Oligonucleotides capable of hybridizing toisolated nucleic acid sequences which include the polymorphism areprovided. It is within the skill of the art to design assays withexperimentally determined stringency to discriminate between the allelicstates of the polymorphisms presented herein. Exemplary assays includeSouthern blots, Northern blots, microarrays, in situ hybridization, andother methods of polymorphism detection based on hybridization Exemplaryoligonucleotides for use in hybridization-based SNP detection areprovided in Table 8. These oligonucleotides can be detectably labeledwith radioactive labels, fluorophores, or other chemiluminescent meansto facilitate detection of hybridization to samples of genomic oramplified nucleic acids derived from one or more soybean plants usingmethods known in the art.

TABLE 8 Oligonucleotide Hybridization Probes Marker SEQ SNP SEQ MarkerID Position Hybridization Probe ID NS0122151 82 62 CCTTGCAAGTCATGCT 201NS0122151 82 62 CCTTGCATGTCATGCT 202 NS0125096 83 139 AAGTTTATGATTTGAA203 NS0125096 83 139 AAGTTTAAGATTTGAA 204 NS0120948 84 109ATTCTTCAGCATGATC 205 NS0120948 84 109 ATTCTTCTGCATGATC 206 NS0092748 100289 TACCTCTAAAACTTGT 207 NS0092748 100 289 TACCTCTTAAACTTGT 208NS0118907 111 449 CTCCAACCTATGATTG 209 NS0118907 111 449CTCCAACATATGATTG 210 NS0092589 117 126 AGCCATCACAAGGAAA 211 NS0092589117 126 AGCCATCATAAGGAAA 212 NS0100457 119 34 TTGGTCCTGCCGGTAA 213NS0100457 119 34 TTGGTCCCGCCGGTAA 214 NS0116259 120 216 TGATAATGACTCCTGA215 NS0116259 120 216 TGATAATAACTCCTGA 216

Example 5 Oligonucleotide Probes Useful for Detecting Soybean Plantswith Aphid Resistance Loci by Single Base Extension Methods

Oligonucleotides can also be used to detect or type the polymorphismsassociated with Soybean aphid resistance disclosed herein by single baseextension (SBE)-based SNP detection methods. Exemplary oligonucleotidesfor use in SBE-based SNP detection are provided in Table 9. SBE methodsare based on extension of a nucleotide primer that is hybridized tosequences adjacent to a polymorphism to incorporate a detectablenucleotide residue upon extension of the primer. It is also anticipatedthat the SBE method can use three synthetic oligonucleotides. Two of theoligonucleotides serve as PCR primers and are complementary to thesequence of the locus which flanks a region containing the polymorphismto be assayed. Exemplary PCR primers that can be used to typepolymorphisms disclosed in this invention are provided in Table 7 in thecolumns labeled “Forward Primer SEQ ID” and “Reverse Primer SEQ ID”.Following amplification of the region containing the polymorphism, thePCR product is hybridized with an extension primer which anneals to theamplified DNA adjacent to the polymorphism. DNA polymerase and twodifferentially labeled dideoxynucleoside triphosphates are thenprovided. If the polymorphism is present on the template, one of thelabeled dideoxynucleoside triphosphates can be added to the primer in asingle base chain extension. The allele present is then inferred bydetermining which of the two differential labels was added to theextension primer. Homozygous samples will result in only one of the twolabeled bases being incorporated and thus only one of the two labelswill be detected. Heterozygous samples have both alleles present, andwill thus direct incorporation of both labels (into different moleculesof the extension primer) and thus both labels will be detected.

TABLE 9 Probes (extension primers) for Single Base Extension (SBE)assays. Marker SEQ SNP SEQ Marker ID Position Probe (SBE) ID NS012215182 62 AGTAGTTACTCCCTTGC 217 NS0125096 83 139 TTCCAAAACTCAAGTTT 218NS0120948 84 109 GTCTGATTAATATTCTT 219 NS0092748 100 289GCATTCCTCAATACCTC 220 NS0118907 111 449 AAAGAGAAAAGCTCCAA 221 NS0092589117 126 GGCCAATAAAAAGCCAT 222 NS0100457 119 34 GGGAGCTAGATTTGGTC 223NS0116259 120 216 TTTAATCAACATGATAA 224

Example 6 Confirmation of Selected Aphid Resistance Alleles

Forty soybean breeding lines were screened for antibiosis in ano-choices field study. Ten plants of each breeding line were planted ina plot. Individual plots were covered by a small insect cage. The cageswere inoculated soybean aphids when the soybean plants reached the VIstage. The plants were rated weekly as described in Table 1. The plantswere assigned a damage index (DI) each week. Table 8 listed the averageDI rating for soybean lines with various aphid resistance alleles.

In addition, the forty lines were screened for antibiosis in a no-choicegreenhouse study. Several plants of each of the forty soybean breedinglines were cultivated in a greenhouse. Leaves were excised from eachplant and inoculated with 2 soybean aphids in closed container. Thenumber of aphids on each leaf was counted after seven days. Table 10listed the average number of aphids on soybean lines with various aphidresistance alleles.

TABLE 10 Confirmation of aphid resistance alleles 1, 16, 23, and 28.Soybean lines were screened for antibiosis to soybean aphid in no-choicegreenhouse and field experiments. Homozygous for Resistance AlleleHomozygous for Susceptible Allele Aphid Field Field Resistance No.Greenhouse Damage No. Greenhouse Damage Locus Plants No. aphids IndexPlants No. aphids Index 1 21 35 36 18 49 88 16 14 38 90 26 45 95 1 + 169 33 32 13 49 88 23 12 45 94 28 48 93 1 + 23 9 31 34 15 50 92 1 + 28 628.1 35.9 11 45 95 1 + 16 + 23 6 30 41 10 48 93 1 + 23 + 28 4 29 43 1041 92 1 + 16 + 28 4 29 43 6 47 93 1 + 16 + 23 + 28 3 26 47 3 46 94 FieldCheck No. Greenhouse Damage Varieties Plants No. aphids Index CNS 2528.2 100.0 PI594427C 19 20.8 25.0 Jackson 25 32.4 21.7 Dowling 9 20.8 —Williams 16 59.4375 —

Aphid resistance allele 1 conferred aphids resistance in both theno-choice field and greenhouse tests. In addition, aphid resistancelocus 16 conferred resistance in no-choice field tests. Moreover, aphidresistance allele 23 enhanced the aphid resistance conferred by aphidresistance allele 1. Similarly, aphid resistance allele 28 enhanced theaphid resistance conferred by aphid resistance allele 1. Soybean plantwith three aphid resistance alleles had a higher level of aphidresistance compared soybean plants with one or two resistance alleles.Furthermore, soybean plants with four aphid resistance alleles possessedthe highest level of aphids resistance.

Host plant resistance management methods may fall into three categories:(1) deploying single resistance alleles sequentially, (2) deployingmultiple varieties with difference single resistance alleles through aseed mixtures or crop rotation, (3) stacking or combining differentresistance allele into a single soybean line. Soybean plants may be bredfor aphid resistance alleles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 singly or incombination depending on the aphid pressure of the geographic region andhost plant resistance management plan.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit, scope and concept of the appended claims.

1. A method of introgressing an allele into a soybean plant comprising:A) Providing a population of soybean plants, B) Genotyping at least onesoybean plant in the population with respect to a soybean genomicnucleic acid marker selected from the group consisting of SEQ ID NO: 81,SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, wherein said nucleicacid markers are indicative of an allele conferring aphid resistance ina soybean plant, C) Selecting at least one soybean plant from thepopulation based upon the presence of at least one of said genomicnucleic acid markers.
 2. The method according to claim 1, wherein saidselected soybean plants exhibit at least partial resistance to aphids.3. The method according to claim 1, wherein said selected soybean plantsexhibit at least substantial resistance to aphids.
 4. The method ofclaim 1, wherein genotyping further comprises an assay which is selectedfrom the group consisting of single base extension (SBE),allele-specific primer extension sequencing (ASPE), DNA sequencing, RNAsequencing, micro-array based analyses, universal PCR, allele specificextension, hybridization, mass spectrometry, ligation,extension-ligation, and Flap-Endonuclease -mediated assays.
 5. Themethod of claim 1 wherein the soybean genomic nucleic acid marker isselected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 82.6. A method of introgressing an allele into a soybean plant comprising:A) crossing at least one aphid resistant soybean plant with at least oneaphid sensitive soybean plant in order to form a segregating population,B) screening said segregating population with one or more nucleic acidmarkers to determine if one or more soybean plants from said segregatingpopulation contains an aphid resistant locus comprising SEQ ID NO: 81,SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO:
 84. 7. The method accordingto claim 6, wherein at least one of said one or more nucleic acidmarkers is located within 30 cM of said resistant locus.
 8. The methodaccording to claim 6, wherein at least one of said one or more markersis located within 20 cM of said resistant locus.
 9. The method accordingto claim 6, wherein at least one of said one or more nucleic acidmarkers is located within 2 cM of said resistant locus.
 10. The methodaccording to claim 6, wherein at least one of said one or more nucleicacid markers is located within 1 cM of said resistant locus.
 11. Amethod of identifying an aphid resistance allele in a soybean plantcomprising detecting a locus associated with aphid resistance with a SNPmarker selected from the group consisting of SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO: 83, and SEQ ID NO: 84.